Mouse embryonic stem (ES) cells can self-renew in the presence of leukemia inhibitory factor (LIF). Several essential transcription factors have been identified for the self-renewal of mouse ES cells, including STAT3, Oct-3/4, and Nanog. The molecular mechanism of ES cell self-renewal, however, is not fully understood. In the present study, we identified Eed, a core component of Polycomb repressive complex 2, as a downstream molecule of STAT3 and Oct-3/4. Artificial activation of STAT3 resulted in increased expression of Eed, whereas expression of a dominant negative mutant of STAT3 or suppression of Oct-3/4 expression led to down-regulation of Eed. Reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays revealed that STAT3 and Oct-3/4 directly bind to the promoter region of Eed, suggesting that Eed is a common target molecule of STAT3 and Oct-3/4. We also found that suppression of STAT3, Oct-3/4, or Eed causes induction of differentiation-associated genes as well as loss of Lys(27)-trimethylated histone H3 at the promoter regions of the differentiation-associated genes. Suppression of STAT3 and Oct-3/4 also resulted in the absence of Eed at the promoter regions. These results suggest that STAT3 and Oct-3/4 maintain silencing of differentiation-associated genes through up-regulation of Eed in self-renewing ES cells.