Neural stem cells are self-renewing cells capable of differentiating into all neural lineage cells in vivo and in vitro. In the present study, coordinated induction of midbrain dopaminergic phenotypes in an immortalized multipotent neural stem cell line can be achieved by both overexpression of nuclear receptor Nurr1, and fibroblast growth factor-8 (FGF-8), and sonic hedgehog (Shh) signals. Nurr1 overexpression induces neuronal differentiation and confers competence to respond to extrinsic signals such as Shh and FGF-8 that induce dopaminergic fate in a mouse neural stem cell line. Our findings suggest that immortalized NSCs can serve as an excellent model for understanding mechanisms that regulate specification of ventral midbrain DA neurons and as an unlimited source of DA progenitors for treating Parkinson disease patients by cell replacement.