Activation of the transcription factor STAT3 is important for astrocyte differentiation during neural development. Demethylation of the methyl-CpG dinucleotide in the STAT3 binding site in the promoter of the glial fibrillary acidic protein (GFAP) gene, a marker for astrocytes, was previously shown to be a crucial cue for neural progenitors to express this gene in response to astrogenic signals during brain development. In this study, we analyzed the methylation status of the STAT3 binding site in the GFAP gene promoter during neural cell development from mouse embryonic stem (ES) cells in vitro. The CpG dinucleotide in the STAT3 binding site in the GFAP gene promoter exhibited a high incidence of cytidine-methylation in undifferentiated pluripotent ES cells. The high incidence of methylation of this particular cytidine was maintained in ES cell-derived neuroectoderm-like cells, but it underwent demethylation when the neural lineage cells became competent to express GFAP in response to a STAT3 activation signal. In contrast, hypermethylation of the CpG site was maintained in non-neural cells generated from the same ES cells. Progressive demethylation of the STAT3 binding site in the GFAP gene promoter was also observed in primary embryonic neuroepithelial cells during in vitro culture, whereas non-neural cells maintained hypermethylation of this site even after culture. Taken together, these results demonstrate that the astrocyte gene-specific cytidine-demethylation is programmed when neural progenitors from pluripotent cells are committed to a neural lineage that is capable of producing astrocytes.