The tumor suppressor von Hippel-Lindau protein (pVHL) is critical for cellular molecular oxygen sensing, acting to target degradation of the hypoxia-inducible factor alpha transcription factor subunits under normoxic conditions. We have found that independent of its function in regulating hypoxic response, the VHL gene plays a critical role in embryonic endothelium development through regulation of vascular extracellular matrix assembly. We created mice lacking the VHL gene in endothelial cells; these conditional null mice died at the same stage as homozygous VHL-null mice, with similar vascular developmental defects. These included defective vasculogenesis in the placental labyrinth, a collapsed endocardium, and impaired vessel network patterning. The defects in embryonic vascularization were correlated with a diminished vascular fibronectin deposition in vivo and defective endothelial extracellular fibronectin assembly in vitro. We found that the impaired migration and adhesion of VHL-null endothelial cells can be partially rescued by the addition of back exogenous fibronectin, which indicates that pVHL regulation of fibronectin deposition plays an important functional role in vascular patterning and maintenance of vascular integrity.