AbstractInside-out signaling regulation of the β2-integrin leukocyte function–associated antigen-1 (LFA-1) by different cytoplasmic proteins, including 14-3-3 proteins, is essential for adhesion and migration of immune cells. Here, we identify a new pathway for the regulation of LFA-1 activity by Cbl-b, an adapter molecule and ubiquitin ligase that modulates several signaling pathways. Cbl-b−/− mice displayed increased macrophage recruitment in thioglycollate-induced peritonitis, which was attributed to Cbl-b deficiency in macrophages, as assessed by bone marrow chimera experiments. In vitro, Cbl-b−/− bone marrow–derived mononuclear phagocytes (BMDMs) displayed increased adhesion to endothelial cells. Activation of LFA-1 in Cbl-b–deficient cells was responsible for their increased endothelial adhesion in vitro and peritoneal recruitment in vivo, as the phenotype of Cbl-b deficiency was reversed in Cbl-b−/−LFA-1−/− mice. Consistently, LFA-1–mediated adhesion of BMDM to ICAM-1 but not VLA-4–mediated adhesion to VCAM-1 was enhanced by Cbl-b deficiency. Cbl-b deficiency resulted in increased phosphorylation of T758 in the β2-chain of LFA-1 and thereby in enhanced association of 14-3-3β protein with the β2-chain, leading to activation of LFA-1. Consistently, disruption of the 14-3-3/β2-integrin interaction abrogated the enhanced ICAM-1 adhesion of Cbl-b−/− BMDMs. In conclusion, Cbl-b deficiency activates LFA-1 and LFA-1–mediated inflammatory cell recruitment by stimulating the interaction between the LFA-1 β-chain and 14-3-3 proteins.