The development of midbrain dopaminergic (DA) neurons is a complex process that requires the precise spatial and temporal expression of numerous genes. Here, we report that Ebf3, a transcription factor, plays a critical role in the terminal development of DA neurons. We found a specific upregulation of Ebf3 in Dicer knockout midbrain DA neurons and dynamic patterns of Ebf3 expression during the development of DA neurons. We further demonstrated that the overexpression of Ebf3 at the neural precursor stage of embryonic stem (ES) differentiation induces a significant increase in the number of TH+ DA neurons, whereas the suppression of Ebf3 leads to significant reduction in the development of TH+ DA neurons. Additionally, we found that Ebf3 is a candidate target for miR218 during DA neuronal development, such that the regulation of Ebf3 expression by miR218 controls the terminal differentiation of DA neurons. Thus, our data suggest that complex transcription factor-miRNA regulation is critical for the development of midbrain DA neurons.