Endothelial cells (EC) are important components for vessel formation and hematopoiesis. The proliferation and differentiation of EC are performed under the close influence of hypoxia-inducible factors (HIFs), which are master transcription factors that regulate vasculogenesis and angiogenesis in response to hypoxic stimuli. During early development of embryos, EC are directly involved in hematopoiesis and are known to act as stromal cells, which generate a variety of regulatory factors, including cytokines and growth factors, and maintain adhesive interactions with the hematopoietic cells essential for their survival and function in the microenvironment. Mouse gene-targeting technology provides us with the information that HIFs are crucial for the development of not only EC but also hematopoietic cells. Although we have determined some particular roles of HIF in association with neovascularization and hematopoiesis in the experiments using gene knockout mice, many crucial roles of HIFs in these processes still remain to be elucidated. Because of the complexity of vasculo/angiogenesis and hematopoiesis in vivo, it is very difficult to analyze distinct involvement of each of the HIFs in the regulation of vessel formation and development of hematopoietic cells. In this chapter, we review the role of HIFs in neovascularization and hematopoiesis with special attention to the usefulness of gene knockdown mice of HIF-2alpha (alpha) to analyze the respective roles of HIFs in these complex processes.