Pluripotent or multipotent stem cells isolated from human embryos or adult central nervous system (CNS) may provide new neurons to ameliorate neural disorders. A major obstacle, however, is that the majority of such cells do not differentiate into neurons when grafted into non-neurogenic areas of the adult CNS. Here we report a new in vitro priming procedure that generates a nearly pure population of neurons from fetal human neural stem cells (hNSCs) transplanted into adult rat CNS. Furthermore, the grafted cells differentiated by acquiring a cholinergic phenotype in a region-specific manner. This technology may advance stem cell–based therapy to replace lost neurons in neural injury or neurodegenerative disorders.