Mice carrying a targeted deletion of the signaling portion of the integrin β4 subunit display drastically reduced angiogenesis in response to bFGF in the Matrigel plug assay and to hypoxia in the retinal neovascularization model. Molecular cytology indicates that α6β4 signaling promotes branching of β4⁺ medium- and small-size vessels into β4⁻ microvessels without exerting a direct effect on endothelial cell proliferation or survival. Signaling studies reveal that α6β4 signaling induces endothelial cell migration and invasion by promoting nuclear translocation of P-ERK and NF-κB. Upon subcutaneous implantation of various cancer cells, the mutant mice develop smaller and significantly less vascularized tumors than wild-type controls. These results provide genetic evidence that α6β4 signaling promotes the onset of the invasive phase of pathological angiogenesis and hence identify a novel target for antiangiogenic therapy.