The introduction of genes associated with the fate of dopaminergic (DA) neurons may help to facilitate the differentiation of embryonic stem cells (ESC) to DA neurons. Here we used lentiviral vectors to drive lmx1b, a key transcription factor of dopaminergic neurons development, expression in ESC. Tyrosine hydroxylase-positive neurons were increased from 18% to nearly 50% when exogenous lmx1b was transduced. The enhanced Pitx3, Girk2, Nurr1 and DAT suggested that the increased differentiated neurons were mature A9 DA neurons. The results of [(3)H]dopamine reuptake assay indicated these differentiated neurons were all functional. Our study demonstrated that lmx1b was capable of promoting the differentiation of ESC to DA neurons.