LIM homeodomain transcription factors, Lmx1a and Lmx1b, are required for the development of midbrain dopaminergic (mDA) neurons. Lmx1b is required for the specification and maintenance of mDA neurons, primarily due to its role in isthmic organizer development that is essential for the induction of mDA neurons. Here, we conditionally deleted Lmx1b in the ventral neural tube usingShhCreand found thatLmx1bconditional mutant mouse embryos show no defect in the development and maintenance of mDA neurons. In addition,Dreher(Lmx1a mutant) embryos display only a moderate reduction in the number of mDA neurons, suggesting that the related family member Lmx1b might compensate for Lmx1a function. We therefore generatedLmx1aandLmx1bdouble mutants. Severe loss of mDA neurons occurred inLmx1adr/dr;ShhCre/+;Lmx1bf/fdouble mutants due to essential roles for Lmx1a and Lmx1b in regulating the proliferation and neuronal commitment of mDA progenitors through the expression of Wnt1 and Ngn2, respectively. Lmx1a and Lmx1b also negatively regulateHes1expression and consequently cell cycle exit through activation of p27Kip1expression. In addition, Lmx1a and Lmx1b also regulate the expression of floor plate genes such asCorinandSlit2and specification of postmitotic mDA neurons. These defects were more severe with decreasing gene dosage of Lmx1a and Lmx1b or observed only when all four copies of Lmx1a and Lmx1b genes were inactivated. Together, our results demonstrate that Lmx1a and Lmx1b function cooperatively to regulate proliferation, specification, and differentiation of mDA progenitors, including their floor plate-like properties.