Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:β2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.Keywords:CD8(+) T cells; chemokines; cytotoxic T cells; diapedesis synapse; endothelium; inflammation; membrane protrusions; transcellular; transmigration.