AbstractDNA methylation is known to regulate cell differentiation and neuronal function in vivo. Here we examined whether deficiency of a de novo DNA methyltransferase, Dnmt3a, affects in vitro differentiation of mouse embryonic stem cells (mESCs) to neuronal and glial cell lineages. Early‐passage neural stem cells (NSCs) derived from Dnmt3a‐deficient ESCs exhibited a moderate phenotype in precocious glial differentiation compared with wild‐type counterparts. However, successive passaging to passage 6 (P6), when wild‐type NSCs become gliogenic, revealed a robust phenotype of precocious astrocyte and oligodendrocyte differentiation in Dnmt3a−/− NSCs, consistent with our previous findings in the more severely hypomethylated Dnmt1−/− NSCs. Mass spectrometric analysis revealed that total levels of methylcytosine in Dnmt3a−/− NSCs at P6 were globally hypomethylated. Moreover, the Dnmt3a−/− NSC proliferation rate was significantly increased compared with control from P6 onward. Thus, our work revealed a novel role for Dnmt3a in regulating both the timing of neural cell differentiation and the cell proliferation in the paradigm of mESC‐derived‐NSCs. © 2012 Wiley Periodicals, Inc.