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Human embryonic stem (hES) cells derived from the inner cell mass of the blastocyst propagate by self-renewal and can give rise to all cells of the body. However, they have a tendency to spontaneously differentiate in vitro, an effect which can be abrogated by culture at low, 5% oxygen tensions. This response is mediated by hypoxia inducible factors (HIFs) and in particular HIF-2α. However, the mechanism of this regulation is still unknown. Chromatin immunoprecipitation (ChIP) analysis showed that HIF-2α directly binds to a predicted hypoxia response element (HRE) in the proximal promoter of OCT4, NANOG, SOX2, GLUT1 and eNOS under hypoxic conditions. An increased level of enrichment (P < 0.01) was observed in hES cells cultured at 5% oxygen whereas no significant binding was observed in cells maintained at 20% oxygen. Interestingly, HIF-2α induced an array of histone modifications that are associated with gene transcription within the predicted HRE site for all the genes analysed. ChIP assays showed that the chromatin state is more accessible and transcriptionally active in hES cells cultured under hypoxic conditions. In contrast, a heterochromatin state exists in the HRE of OCT4, SOX2, NANOG, GLUT1 and eNOS in hES cells cultured under 20% oxygen tension. This was also confirmed using pyrosequence analysis which revealed a significant hypomethylation pattern (P < 0.01) in the HRE site within the OCT4 proximal promoter in hES cells cultured at 5% oxygen compared to those maintained at 20% oxygen. hES cells were then used as a model to investigate the mechanisms acquired by resident stem cell populations to maintain an undifferentiate and proliferative state at the site of injury which are characterized by hypoxia and oxidative stress. Interestingly, an enhanced euchromatic state was found when hES cells were exposed to hypoxia/reoxygenation for 72 hours. Surprisingly, this was sustained by HIF-2α which was found significantly enriched within the HRE of all core pluripotency genes but particularly within the NANOG gene promoter. Furthermore, HIF-2α was found responsible of the establishment of a multiprotein complex thereby allowing interaction with an oct-sox cis-regulatory element in the NANOG promoter and sustaining selfrenewal. Thus, these data have uncovered a novel role of HIF-2α as a direct regulator of key transcription factors controlling self-renewal and epigenetic modifications which enhances the regenerative potential of hES cells exposed to hypoxia and reoxygenation.