538 and progenitor cells (NSC) severely disrupts murine brain growth, particularly that of the cerebellum, while a similar KO of c-myc moderately impairs growth [16, 17]. Disrup- tion of either c- or N-myc or both in hematopoietic stem cells (HSC) also alters their normal biological functions, affecting survival and self-renewal [18, 19]. These studies support the hypothesis that every normal cell has a requirement for some minimum level of myc. myc genes also are involved in the production of induced pluripotent stem (iPS) cells (reviewed in [20]) [21–25]. While exogenous Myc is not formally required for the process [26, 27], it dramatically enhances the efficiency and in its absence its function is likely supplanted by endogenous myc. Data suggests that during iPS cell formation, Myc represses differentiation-associated genes [28] and may not have a key role in directly maintaining expression of pluripotency factors. However, in neuroblastoma and other tumors, some pluripotency genes such as lif, lin28b, Klf2, and Klf4 are N-Myc targets for activation, while a subset of these genes is also regulated in NSC by N-Myc [29]. Perhaps because of the importance of myc genes in normal cellular biology and their ability to cause cancer when in excess, cells have evolved systems to maintain normal total cumulative myc RNA and Myc protein levels. These include cross-regulation, redundancy and compensation between the three main myc genes—c-, N-, and L-myc—as well as Myc- trigged apoptosis when in excess. Myc protein stability is also tightly controlled (reviewed in [30]). While desirable for the cell and organism, this interconnectedness has made it more difficult for the biologist to discern the specific roles of individual myc genes through methods such as KO studies. For example, despite fairly ubiquitous expression in the developing brain and some other regions of the embryo, L- myc constitutive KO was reported to have no phenotype at all [31]. One theory is that this lack of apparent phenotype was due to the continued presence of N-myc and perhaps c- myc, which could fulfill the roles of L-myc. In contrast, constitutive KO of either c- or N-myc resulted in midgesta- tional lethality [14, 15, 32], suggesting their overall embryonic functions are to some critical degree unique or their loss cannot be compensated for. Conditional double knockout (DKO) of c- and N-myc in hematopoietic stem cells yields a far more severe phenotype than disruption of either gene alone, suggesting additive or redundant roles [19]. A large degree of redundancy is also supported by the knocking of N-myc into the c-myc locus largely rescuing the loss of c-myc [33]. The prevailing theory is that what is most critical is the total level of all myc gene expression in each cell. During neurogenesis, N-myc plays a particularly impor- tant role in NSC to direct overall brain and particularly cerebellar growth [16, 34], consistent with its fairly widespread expression pattern in the brain throughout Cerebellum (2010) 9:537–547 embryogenesis including very high levels in the developing cerebellum. L-myc is also fairly widely expressed, particu- larly in the early midbrain, which suggests it may function in the brain despite no reported KO phenotype. Although c-myc expression has been less clearly defined, it appears to be more restricted than that of L- and N-myc. In vitro overexpression and c-myc KO studies in rat neurospheres support a crucial role for myc genes overall in NSC function and suggested functional ties to p53/Arf [35], but the relevance for in vivo neurogenesis remained unclear. While no nestin-cre L-myc conditional knockout has been reported, single nestin-cre-driven knockout of c-myc in NSC impairs brain growth, but far less severely than loss of N-myc and with no cerebellar specific phenotype [16, 34]. The molecular basis of the c- and N-myc nestin-cre-driven brain phenotypes is not completely clear, but in the N-myc knockout changes in cyclin D2 and cyclin-dependent kinase inhibitors such as p18INK4C and p27KIP1 were evident suggesting cell cycle regulation plays an important role [16, 36], including in the cerebellum. However, no unbiased global analysis of gene expression has been conducted for myc genes using a loss of function model in the developing brain or specifically in the cerebellum. Upstream of N-myc are several potential signaling pathways including Shh in the cerebellum and in CGNPs [17, 37], while N-myc also appears to be a critical part of a newly defined pathway DLL3 and Notch signaling in which N-Myc protein is targeted by the Huwe1 ubiquitin ligase [38, 39]. To address the endogenous functions of c- and N-myc in cerebellar development, we created a nervous system specific double c- and N-myc KO mouse by crossing doubly homozygously myc floxed mice with nestin-cre, which is expressed specifically in NSC and at very high levels in CGNP and in NSC of the cerebellum neuro- epithelium. The myc DKO mice have a nervous system phenotype much more severe than either single myc KO alone, which is ultimately lethal. In terms of mechanisms, we found evidence that Myc regulates transcriptional programs including that of metabolic genes essential for normal stem and precursor fate during cerebellar genesis. Methods and Methods Animals Breeding pairs of nestin-Cre− N-myc(FL/FL) c-myc(FL/FL) and nestin-Cre+ N-myc(FL/WT) c-myc(FL/FL) [10] were set up to generate the four genotypes Nestin-Cre− N-myc(FL/FL) c- myc(FL/FL), Nestin-Cre− N-myc(FL/WT) c-myc(FL/FL), Nestin- Cre+ N-myc(FL/FL) c-myc(FL/FL), and nestin-Cre+ N-myc(FL/WT) c-myc(FL/FL). Genotypes of animals were determined via polymerase chain reaction (PCR) using primer sets for Cre,