Precise spatiotemporal regulation of leukocyteextravasationis key for generating an efficient immune response to injury or infection. The integrinsLFA-1(CD11a/CD18) andMac-1(CD11b/CD18) play overlapping roles inneutrophilmigration because they bind the same as well as different ligands in response to extracellular signaling. Using two-photon intravital imaging and transmission electron microscopy, we observed the existence of preferred sites forneutrophilentrance into the endothelial cell monolayer and exit from the basement membrane and pericyte sheath duringneutrophilextravasation, namely,hotspotsI andII, by elucidating distinctive roles ofLFA-1andMac-1. To penetrate the vascular endothelium, neutrophils must first penetrate the endothelial cell layer through hotspot I (i.e., the point of entry into the endothelium). Neutrophils frequently remain in the space between the endothelial cell layer and the basement membrane for a prolonged period (>20 min). Subsequently, neutrophils penetrate the basement membrane and pericyte sheath at hotspotII, which is the final stage of exiting the vascular endothelium. To further investigate the roles ofLFA-1andMac-1, we newly generatedLFA-1FRET (CD11a-YFP/CD18-CFP) mice andMac-1FRET (CD11b-YFP/CD18-CFP) mice. Using both FRET mice, we were able to determine thatLFA-1andMac-1distinctlyregulatetheneutrophilextravasationcascade. Our data suggest that the vascular endothelium functions as a double-layered barrier in the steps ofneutrophilextravasation. We propose that the harmonized regulation ofneutrophilpenetration through the endothelium viahotspotsI andIImay be critical for vascular homeostasis during inflammation.