Delta-like 1 (Dlk1), a member of the Delta/Notch protein family, is expressed in the mouse ventral midbrain (VM) as early as embryonic day 11.5 (E11.5) followed by exclusive expression in tyrosine 3-monooxygenase (TH) positive neurons from E12.5 onwards. To further elucidate the yet unknown function of Dlk1 in VM neuron development, we investigated the effect of soluble Dlk1 protein as well as the intrinsic Dlk1 function in the course of VM progenitor expansion and dopaminergic (DA) neuron differentiation in vitro. Dlk1 treatment during expansion increased DA progenitor proliferation and the proportion of NR4A2+ neurons expressing TH after differentiation, whereas Dlk1 treatment during the course of DA precursor differentiation did not alter TH+ neuron counts. In contrast, silencing of endogenously expressed Dlk1 prior to DA precursor differentiation partially prevented the expression of DA neuron markers, which was not accompanied with alteration of overall or local proliferation. Due to the latter finding in combination with the absence of Dlk1 negative DA neurons in differentiated cultures, we suggest that Dlk1 expression might have a permissive effect on DA neuron differentiation in vitro. The study presented here is the first publication identifying Dlk1 effects on ventral midbrain-derived DA precursor differentiation.