Protocols used for generation of mesencephalic dopamine (mesDA) neurons from stem cells, or fetal brain tissue, invariably result in cell preparations that are highly mixed in composition, containing mesDA neuron precursors in various states of fate commitment and differentiation. For further optimisation and refinement of these procedures it is essential to determine the optimal stage of development and phenotypic characteristics of cells used for grafting. We have used fluorescence-activated cell sorting procedures to isolate mesDA precursors in defined stages of differentiation from mouse ventral mesencephalon (VM), at embryonic day 10.5 (E10.5), when the mesDA neuron domain consists of proliferative radial glia-like cells expressing the mesDA neuron determinant Lmx1a and the floorplate marker Corin, and at E12.5, when the VM has expanded to comprise a mixture of proliferative progenitors, neuroblasts and young neurons. The sorted cells were transplanted to the striatum of 6-hydroxydopamine-lesioned rats. Results show that the Lmx1a/Corin-expressing ventricular zone progenitors, which are the source of mesDA neurons in grafts from E10.5 VM, had lost this capacity at E12.5. At this later stage all transplantable mesDA precursors resided in the intermediate zone as postmitotic Nurr1-expressing neuroblasts. The more differentiated, TH-expressing cells survived sorting and transplantation poorly. We also provide evidence that, during early mesDA neurogenesis, the progenitors for nigral mesDA neurons segregate to lateral parts of the Lmx1a-expressing domain and can be selectively isolated based on their level of Corin expression. These results have implications for current efforts to develop well-characterized stem cell-derived mesDA progenitor cell preparations for cell therapy.