In the presented work, we showed that both, VSV-G and LCMV-GP pseudotyped lentiviruses efficiently transduced human glioma cells in vitro and in vivo, whereas gammaretroviral transduction was inefficient. The gene transfer to glioma cells was efficient for both lentiviral pseudotyped vector types. However, it was more specific using LCMV-GP pseudotyped vectors, as VSV-G pseudotypes also transduced host brain cells in invasive areas. Analysis of transduced tumor cells revealed that both lentiviral vectors targeted CD133-positive as well as CD133negative cancer cells. Furthermore, transduced glioblastoma cells expressed the stem cell markers nestin and SOX2. Importantly, when evaluated for therapeutic application using HSV-1-tk as a transgene, both lentiviral vectors mediated complete tumor regression on MRI, resulting in a highly significant survival benefit (p,0.001) compared to the control groups.