The hypoxia-inducible factors have recently been identified as critical regulators of angiogenic–osteogenic coupling. Mice overexpressing HIFα subunits in osteoblasts produce abundant VEGF and develop extremely dense, highly vascularized long bones. In this study, we investigated the individual contributions of Hif-1α and Hif-2α in angiogenesis and osteogenesis by individually disrupting each Hifα gene in osteoblasts using the Cre-loxP method. Mice lacking Hif-1α demonstrated markedly decreased trabecular bone volume, reduced bone formation rate, and altered cortical bone architecture. By contrast, mice lacking Hif-2α had only a modest decrease in trabecular bone volume. Interestingly, long bone blood vessel development measured by angiography was decreased by a similar degree in both ΔHif-1α and ΔHif-2α mice suggesting a common role for these Hifα subunits in skeletal angiogenesis. In agreement with this idea, osteoblasts lacking either Hif-1α or Hif-2α had profound reductions in VEGF mRNA expression but only the loss of Hif-1α impaired osteoblast proliferation. These findings indicate that expression of both Hif-1α and Hif-2α by osteoblasts is required for long bone development. We propose that both Hif-1α and Hif-2α function through cell non-autonomous modes to promote vascularization of bone and that Hif-1α also promotes bone formation by exerting direct actions on the osteoblast. J. Cell. Biochem. 109: 196–204, 2010. © 2009 Wiley-Liss, Inc.