Figure 5(a) FACS-sorted HE cells were subcultured with or without TSA (60nM). Subculture day 3 CD43 MFI levels ± SEM and a representative CD43 histogram are shown (n=3 biological replicates 1-2 technical replicates each, paired t test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (b) Dot plots showing gene expression levels of LSD1, GFI1 and GFI1B detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (c) FACS-sorted HE cells were subcultured with TCP (300 nM), UK5099 (10 µM) or both and day 3 CD43+GPA+ cell frequencies ± SEM relative to the control are shown (n=4 biological replicates, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (d) HE cells were transduced with shScrambled (shScr) or shLSD1 with or without UK5099 (10 µM) the day after the sort and day 3 CD43+/GPA+ cell frequencies ± SEM relative to shScr are presented (n=3 biological replicates 1-2 technical replicates each, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (e) Acetate can be directly converted to acetyl-coA by ACSS2 (inhibitor: ACSS2i). Acetyl-coA is the precursor of acetylation marks, transferred onto histones via histone acetyltransferases (HATs, inhibitor: C646). (f-g) FACS-sorted HE cells were subcultured with ACSS2i (5 µM), DCA (3 mM), or both (n=5 biological replicates, one-way ANOVA test) (f) or with C646 (10 µM), DCA (3 mM), or both (n=3 biological replicates, one-way ANOVA test) (g) and day 6 CD43+CD45+ cell frequencies ± SEM are shown. Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (h) FACS-sorted HE cells were subcultured with or without DCA (3 mM) for 2 days on coverslips. Staining intensities of H3K9 acetylation and H4K5, 8, 12, 16 acetylation were assessed by confocal microscopy imaging and fold change ± SEM compared to the control is shown (n=3 biological replicates). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (i) Acetyl-coA can be a precursor for lipid biosynthesis via ACC (inhibitor: CP-640186 or CP) or for the mevalonate pathway/cholesterol biosynthesis via HMGCR (inhibitor: Atorvastatin or Ato). (j) FACS-sorted HE cells were subcultured with CP (5 µM), DCA (3 mM) or both and day 6 CD43+CD45+ cell frequencies ± SEM relative to the control are shown (n=4 biological replicates, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (k) Cholesterol content ± SEM in HE cells was measured by confocal microscopy at day 2 of DCA (3 mM) treatment by filipin III staining (n=3 biological replicates, paired t test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (l) Cholesterol content ± SEM in HE cells was assessed by flow cytometry after intracellular staining with filipin III at day 3 of DCA treatment (n=3 biological replicates, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (m) FACS-sorted HE cells were subcultured with Ato (0.5 µM), DCA (3 mM) or both and day 3 CD43+CD45+ cell frequencies ± SEM relative to the control are shown (n=3 biological replicates for control/DCA, n=2 biological replicates with 2 technical replicates for Ato/Ato+DCA, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (n) Dot plots showing gene expression levels of NOTCH1, JAG1 and HES1 detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity). (o) Notch1 expression (MFI ± SEM) in HE-derived CD43+CD45+ cells at day 3 of subculture with or without DCA (3 mM) was assessed by flow cytometry (n=2 biological replicates with 1-2 technical replicates, unpaired t test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (p) Glycolysis is essential for hematopoietic differentiation of HE cells and inhibiting pyruvate entry into mitochondria (via UK5099 or shMPC1/2) favors a primitive erythroid fate. Increasing pyruvate flux into mitochondria (via DCA or shPDK4) amplifies acetyl-coA production which fuels cholesterol biosynthesis and promotes definitive hematopoietic differentiation of HE cells.