Leukocyte transmigration is a key event in host defense. As well as delivering leukocytes to sites of inflammation, this response also has an important role in immunity by regulating the responsiveness and behavior of leukocytes in the extravascular tissue. Recent evidence suggests that these events are associated with phenotypic and functional changes in migrating leukocytes mediated by signaling and transcriptional events triggered by the molecular interactions involved in leukocyte transendothelial cell migration. Transfer of membrane proteins from endothelial cells to migrating leukocytes and interaction of leukocytes with components of the perivascular basement membrane might also contribute to this effect. This Review will discuss the characteristics, potential mechanisms and the relevance of transmigration-induced change in leukocyte phenotype and responsiveness both within physiological and pathological scenarios.