Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α. The HIF-1α and HIF-2α subunits are structurally similar in their DNA binding and dimerization domains but differ in their transactivation domains, implying they may have unique target genes and require distinct transcriptional cofactors. Our previous results demonstrated that HIF-1α and HIF-2α regulate distinct target genes. Here, we report that HIF-2α is not transcriptionally active in embryonic stem (ES) cells, as well as possible inhibition by a HIF-2α-specific transcriptional repressor. Using DNA microarray analysis of hypoxia-inducible genes in wild-type (WT), Hif-1α^−/−, and Hif-2α^−/− ES cells, we show that HIF-1α induces a large number of both confirmed and novel hypoxia-inducible genes, while HIF-2α does not activate any of its previously described targets. We further demonstrate that inhibition of HIF-2α function occurs at the level of transcription cofactor recruitment to endogenous target gene promoters. Overexpression of WT and, notably, a DNA-binding-defective HIF-2α mutant restores endogenous HIF-2α protein activity, suggesting that ES cells express a HIF-2α-specific corepressor that can be titrated by overexpressed HIF-2α protein. HIF-2α repression may explain why patients with mutations in the VHL tumor suppressor gene display cancerous lesions in specific tissue types.