Abstract only The proneural basic‐helix‐loop‐helix (bHLH) transcription factors Ascl1 and Neurog2 direct the differentiation of specific populations of neurons by regulating the transcription of specific sets of genes during embryonic development. Here we describe the generation of stable P19 embryonic carcinoma cells expressing comparable levels of Ascl1 and Neurog2 mRNA in a tetracycline inducible system and the resulting neuronal differentiation of these cell lines in response to doxycycline. Microarray analysis of these cell lines showed that Ascl1 and Neurog2 overlap in their gene regulation but each bHLH protein also regulated distinct genes. We demonstrated that DNA methylation accompanied repression of the Oct4 gene by both Ascl1 and Neurog2, although DNA demethylation was not observed for any genes induced by either Ascl1 or Neurog2. While the ID1 gene showed preferential induction by Ascl1 over Neurog2, the induction of ID1 reporter constructs was dependent on distinct sets of E‐boxes within the ID1 proximal promoter. Finally, the induction of the Ppp1r14a gene was specifically induced by Neurog2 and this induction was at least partially due to preferences of Neurog2 for distinct canonical E‐box sequences within the Ppp1r14a promoter. Together, these results suggest that further complexities in the mechanisms by which Ascl1 and Neurog2 regulate gene expression during neuronal differentiation.