The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic “knock-in” strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1α with HIF-2α results in expanded expression of HIF-2α-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2α, but not HIF-1α, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2α KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2α severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2α-specific target gene and indicate that HIF-2α can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2α's tumor promoting activity.