The adult mammalian forebrain subependyma contains neural stem cells (NSCs) capable of self-renewal and multilineage differentiation. The in vivo identification of NSCs has not been definitively addressed using a loss of function approach. Using a transgenic mouse expressing herpes-simplex virus thymidine kinase from the glial fibrillary acidic protein (GFAP) promotor, we have selectively killed dividing GFAP-positive cells in the presence of ganciclovir (GCV) and shown a > 95% loss in the numbers of NSCs, as assayed by the formation of clonally derived neurospheres in vitro. This loss is seen following 3 days of GCV exposure in vivo or in vitro only and cannot be rescued by coculturing with pure astrocyte populations or control (green fluorescent protein-expressing) subependymal cells. Exposure to GCV in vitro has no effect on adult retinal stem cells hence, we conclude that adult forebrain NSCs comprise a subpopulation of the GFAP-positive cells within the subependyma.