Objective— β3-integrin deficiency has been implicated in increasing levels of Flk-1 expression on endothelial cells and enhancing vascular endothelial growth factor (VEGF)-induced angiogenesis. We determined the role of β3-integrin in mediating VEGF-A–induced blood vessel permeability through Flk-1. Methods and Results— Using the Miles assay, we demonstrated that VEGF-A–induced plasma leakage was enhanced in β3-null mice when compared with wild-type controls. This was not caused by any changes in blood vessel structure (as detected by light or electron microscopy) or by changes in endothelial cell–cell adhesion proteins (as determined by Western blot analysis, flow cytometry, and immunofluorescence). Circulating levels of VEGF, baseline blood vessel leakage, and leakage in response to an acute inflammatory stimulus were identical in wild-type and β3-null mice. However, VEGF-A–induced leakage was abolished in β3-null mice by the inhibition of Flk-1, indicating that the elevated levels of Flk-1 on β3-null endothelial cells enhance VEGF-A–induced permeability. Conclusions— β3-integrin–deficiency increases the sensitivity of endothelial cells to VEGF-A by elevating Flk-1 expression and, as a consequence, enhances VEGF-A–mediated permeability.