Transcription factors and epigenetic modulators are involved in the maintenance of self-renewal in embryonic stem (ES) cells. Here, we demonstrate the existence of a regulatory loop in ES cells between Sox2, an indispensable transcription factor for self-renewal, and embryonic ectoderm development (Eed), an epigenetic modulator regulating histone methylation. We found that Sox2 and Eed positively regulate each other's expression. Interestingly, Sox2 overexpression suppressed the induction of differentiation-associated genes in Eed-deficient ES cells without restoring histone methylation. This Sox2-mediated suppression was prevented by knockdown of the histone acetyltransferase (HAT), Tip60 or Elp3, and Sox2 stimulated expression of these HATs. Furthermore, forced expression of either HAT resulted in repression of differentiation-associated genes in Eed-deficient cells. These results suggest that Sox2 overcame the phenotype of Eed-deficient ES cells by promoting histone acetylation. We also found that knockout of Eed and knockdown of these HATs synergistically enhanced the upregulation of differentiation-associated genes in ES cells. Taken together, our results suggest that the Eed/Sox2 regulatory loop contributes to the maintenance of self-renewal in ES cells by controlling histone methylation and acetylation.