Recent studies of mouse mutant aphakia have implicated the homeobox gene Pitx3 in the survival of substantia nigra dopaminergic neurons, the degeneration of which causes Parkinson’s disease. To directly investigate a role for Pitx3 in midbrain DA neuron development, we have analyzed a line of Pitx3-null mice that also carry an eGFP reporter under the control of the endogenous Pitx3 promoter. We show that the lack of Pitx3 resulted in a loss of nascent substantia nigra dopaminergic neurons at the beginning of their final differentiation. Pitx3 deficiency also caused a loss of tyrosine hydroxylase (TH) expression specifically in the substantia nigra neurons. Therefore, our study provides the first direct evidence that the aphakia allele of Pitx3 is a hypomorph and that Pitx3 is required for the regulation of TH expression in midbrain dopaminergic neurons as well as the generation and/or maintenance of these cells. Furthermore, using the targeted GFP reporter as a midbrain dopaminergic lineage marker, we have identified previously unrecognised ontogenetically distinct subpopulations of dopaminergic cells within the ventral midbrain based on their temporal and topographical expression of Pitx3 and TH. Such an expression pattern may provide the molecular basis for the specific dependence of substantia nigra DA neurons on Pitx3.