Widely coexpressed Src family kinase (SFK) members Src, Fyn and Yes are involved in various cellular events, often acting downstream of receptor tyrosine kinases, such as vascular endothelial growth factor (VEGF) receptors. They are well known for their functional redundancy; any unique features remain largely undefined. Utilizing RNA interference, we have selectively knocked down Src, Fyn and Yes in human retinal microvascular endothelial cells (HRMECs). Cells with single SFK knockdown showed that all three kinases were required for VEGF mitogenic signaling. VEGF-induced cell migration was significantly increased in Fyn-deficient cells and decreased in Yes-deficient cells. Selective interference of Fyn, but not Src or Yes, impaired VEGF-induced tube formation in HRMECs. Cells in which all three SFKs were targeted showed significant inhibition of all three cellular events. In addition, interference of Src, Fyn and Yes did not affect the anti-apoptotic effect of VEGF in HRMECs, as determined by DNA fragmentation analysis. These results provide direct evidence that Src, Fyn and Yes maintain distinct properties in the regulation of VEGF-mediated endothelial cell events.