Glioblastomas exhibit a hierarchical cellular organization, suggesting that they are driven by neoplastic stem cells that retain partial yet abnormal differentiation potential. Here, we show that a large subset of patient-derived glioblastoma stem cells (GSCs) express high levels of Achaete-scute homolog 1 (ASCL1), a proneural transcription factor involved in normal neurogenesis. ASCL1^hi GSCs exhibit a latent capacity for terminal neuronal differentiation in response to inhibition of Notch signaling, whereas ASCL1^lo GSCs do not. Increasing ASCL1 levels in ASCL1^lo GSCs restores neuronal lineage potential, promotes terminal differentiation, and attenuates tumorigenicity. ASCL1 mediates these effects by functioning as a pioneer factor at closed chromatin, opening new sites to activate a neurogenic gene expression program. Directing GSCs toward terminal differentiation may provide therapeutic applications for a subset of GBM patients and strongly supports efforts to restore differentiation potential in GBM and other cancers.