Glioblastoma (GBM) is the most common and aggressive form of primary brain cancer with extremely poor prognosis. New approaches for improved treatment of this disease are desperately needed. Accumulating evidence suggests that the lack of effective treatment for this disease is because the existing regimens cannot effectively eliminate glioblastoma stem cells (GSCs), the root of GBM development. One promising approach to eliminating GSCs is to induce GSC differentiation. Unfortunately, the development of differentiation therapy for GBM has been limited by our poor understanding of the biology of GSC differentiation. In this application, we propose to characterize genetic regulation of GSC differentiation and validate genes that can be targeted for effective GBM differentiation therapy. As preliminary work, we identified a group of genes, which potentially regulate GSC differentiation toward specific lineages through a combination of RNAi screening and single-cell RNA sequencing approach. In this application, we will characterize lead GSC differentiation regulatory candidate genes in Aim 1, and to determine the therapeutic potential of selected genes for GSC differentiation therapy in Aim 2. Success of this project will lead to identification of novel GSC differentiation regulators that can be potentially targeted for improved treatment of GBM.