Cytotoxic T lymphocytes (CTLs) kill target cells via fusion of lytic granules (LGs) at the immunological synapse (IS). Soluble NSF-attachment protein receptors (SNAREs) function as executors of exocytosis. The importance of SNAREs in CTL function is evident in the form of familial hemophagocytic lymphohistiocytosis (FHL) type 4 which is caused by mutations in Syntaxin11 (Stx11), a Qa-SNARE protein. Here, we investigate the molecular mechanism of Stx11 function in primary human effector CTLs with high temporal and spatial resolution. Downregulation of endogenous Stx11 resulted in a complete inhibition of LG fusion that was paralleled by a reduction in LG dwell time at the IS. Dual color evanescent wave imaging suggested a sequential process, in which first Stx11 is transported to the IS through a subpopulation of recycling endosomes. The resulting Stx11 clusters at the IS then serve as a platform to mediate fusion of arriving LGs. We conclude that Stx11 functions as a t-SNARE for the final fusion of LG at the IS, explaining the severe phenotype of FHL4 on a molecular level. This article is protected by copyright. All rights reserved.