The extravasation of leukocytes is a critical step during inflammation which requires the localized opening of the endothelial barrier. This process is initiated by the close interaction of leukocytes with various adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) on the surface of endothelial cells. It is still unclear how these initial processes induce downstream signaling events resulting in the opening of inter-endothelial junctions to allow leukocyte diapedesis. Here we show that mechanical forces induced by leukocyte-induced clustering of ICAM-1 and fluid shear stress exerted by the flowing blood synergistically activate the mechanosensitive cation channel PIEZO1 in endothelial cells. In human and mouse endothelial cells exposed to low flow, PIEZO1 mediates leukocyte-induced increases in Ca2+i and activation of downstream signaling events including phosphorylation of SRC, PYK2 and myosin light chain (MLC) leading to endothelial barrier opening. Mice with endothelium-specific loss of Piezo1 show decreased leukocyte extravasation in different inflammation models. We found that actin polymerization and actomyosin contraction induced by ICAM-1 clustering synergistically with fluid shear stress increase endothelial plasma membrane tension to activate PIEZO1. Our data reveal a mechanism by which leukocytes and the hemodynamic microenvironment synergize to mechanically activate endothelial PIEZO1 and subsequent downstream signaling to initiate leukocyte diapedesis.