AbstractThe growth of glioblastoma (GBM), one of the deadliest adult cancers, is fuelled by a subpopulation of stem/progenitor cells, which are thought to be the source of resistance and relapse after treatment. Re-engagement of a latent capacity of these cells to re-enter a trajectory resulting in cell differentiation is a potential new therapeutic approach for this devastating disease. ASCL1, a proneural transcription factor, plays a key role in normal brain development and is also expressed in a subset of GBM cells, but fails to engage a full differentiation programme in this context. Here, we investigated the barriers to ASCL1-driven differentiation in GBM stem cells. We see that ASCL1 is highly phosphorylated in GBM stem cells where its expression is compatible with cell proliferation. However, overexpression of a form of ASCL1 that cannot be phosphorylated on Serine–Proline sites drives GBM cells down a neuronal lineage and out of cell cycle more efficiently than its wild-type counterpart, an effect further enhanced by deletion of the inhibitor of differentiation ID2, indicating mechanisms to reverse the block to GBM cell differentiation.