Solid tumors often contain regions with insufficient oxygen delivery, a condition called hypoxia. Tumor hypoxia is an independent prognostic factor significantly correlated with advanced stages of malignancy, increased resistance to conventional therapy, and reduced disease-free survival. Hypoxic tumor cells exhibit poorly differentiated phenotypes resembling stem or progenitor cells. Studies have shown that hypoxia can inhibit tumor cell differentiation and promote maintenance of cancer stem cells. In addition, hypoxia also blocks the differentiation of mesenchymal stem/progenitor cells, a potential source of tumor-associated stromal cells. Therefore, hypoxia may play a critical role during the evolution of the tumor stromal microenvironment and formation of the putative cancer stem cell niches. Conceptually, hypoxia may help create a microenvironment enriched both in poorly differentiated tumor cells and in undifferentiated stromal cells. Such an undifferentiated hypoxic microenvironment may provide essential cellular interactions and environmental signals for the preferential maintenance of cancer stem cells. This review will discuss the hypoxia-regulated stem cell pathways and their roles in the maintenance of cancer stem cell functions.