The basic helix-loop-helix transcription factor Hand2 is induced by bone morphogenetic proteins (BMPs) in neural crest-derived precursor cells during the early stage of development of the autonomic nervous system (ANS). Previous studies showed that Hand2 was essential for the ANS differentiation. However, regulatory mechanism of pluripotent genes has not been elucidated in ANS differentiation. Here, we show that Hand2 regulated nanog expression in ANS differentiation. Our studies demonstrated that the forced expression of Hand2 promoted the ANS differentiation program in P19 embryonal carcinoma (EC) cells without aggregation. Furthermore, our results suggested that Hand2 bound to the promoter of nanog, a gene required for embryonic stem cells self-renewal, and suppressed nanog expression after Hand2 induction. The rapid downregulation of nanog mRNA during ANS differentiation correlated with the Hand2 transcriptional activity and nanog promoter methylation. These findings are evidence for a presence of the novel regulatory mechanism of nanog in ANS differentiation.