Small extracellular vesicles (sEVs) derived from cytotoxic T lymphocytes (CTLs) are emerging as potential mediators of antitumor immunity; however, their subcellular origins and functional properties remain incompletely defined. In this study, we investigated the intracellular routes and cytotoxic potential of CTL-derived exosomes. Using correlative light and electron microscopy, we discovered that CTL-derived exosomes originate from both classical multivesicular bodies (MVBs) and the recently identified multi core granules (MCGs). Through total internal reflection fluorescence microscopy, we demonstrated that, in contrast to MVB-derived exosomes, MCG-derived exosomes are released at the immunological synapse in a stimulus-dependent manner. To enable functional characterization, we developed a scalable primary cell culture method for the isolation of high-purity exosomes. Super-resolution microscopy revealed significant heterogeneity in exosome size and tetraspanin composition. Notably, MCG-derived exosomes exhibited fivefold higher cytotoxic activity than MVB-derived exosomes, inducing apoptosis in tumor cells via a caspase 3-dependent mechanism. These findings reveal that CTLs exploit distinct secretory pathways to release heterogeneous exosome populations with differential cytotoxic capacities, offering new insights into CTL-mediated immune responses and providing a basis for the development of novel exosome-based immunotherapies.