Abstract Neural stem cells (NSCs), either isolated from fetal or adult human brain or derived from induced pluripotent stem cells, are now considered major candidates for in vitro generation of transplantable dopaminergic (DA) neurons and modeling of Parkinson's disease. It is generally thought that in vitro differentiation of neural stem cells into meso-diencephalic dopaminergic neurons, requires recapitulation of dopaminergic differentiation pathway normally occurring in the ventral mesencephalon during embryogenesis. This dopaminergic pathway is partially activated by a combination of the extracellular induction factors Sonic Hedgehog (Shh), Fibroblast Growth Factor 8 (FGF8) and Wnt1 that trigger specific intracellular transcription cascades. In vitro mimicking of these embryonic ventral mesencephalic conditions has been successful for dopaminergic differentiation of embryonic stem cells and ventral mesencephalic NSCs. Dopaminergic differentiation of non-mesencephalic NSCs (nmNSCs), however, is considered arduous. Here we examine whether Shh, FGF8 and Wnt1 can activate typical dopaminergic transcription factors, such as Lmx1a, Msx1 and Otx2 in nmNSCs. We found that Shh, FGF8 and Wnt1 induced the expression of Lmx1a and Otx2 in nmNSCs resulting in the differentiation of up to 39% of the nmNSCs into neurons expressing Pitx3. However, only a low number (∼13%) of these cells became more DA-like neurons also expressing tyrosine hydroxylase (TH). The histone deacetylase (HDAC)-inhibitor trichostatin A combined with Shh, FGF8 and Wnt1 caused orchestrated induction of Lmx1a, Otx2, Msx1 plus the early DA transcription factor En1. Now significantly increased numbers of TH (∼22%) and Pitx3 (∼33%) neurons were observed. Most of these cells coexpressed the DA markers DAT and Vmat2. Taken together, we demonstrate that nmNSCs indeed can be differentiated towards DA-like neurons, but this differentiation is far from complete in comparison to ventral mesencephalic NSCs and embryonic stem cells; most likely, the nmNSCs lack the proper “primed” epigenetic state of these cells for DA differentiation facilitating the induction of DA specific transcription factors.