Cytotoxic T lymphocytes (CTL) can kill tumor cells and cells infected with intracellular pathogens. Their two major killing mechanisms are the degranulation pathway and the Fas-FasL pathway. The degranulation process consists of the release of cytolytic perforin and granzyme molecules that are stored within lysosomal granules. The cytolytic components of granules contain sorting signals within their sequences that act as signals to direct their transport to lysosomes after their biosynthesis. When CTL become stimulated after encountering their target cells, the granules move along microtubules to the contact point with the target cell where a set of vesicle trafficking proteins mediate the specific fusion of the granules with the plasma membrane allowing the release of their cytolytic contents. The Fas-FasL pathway consists of the expression of Fas ligand (FasL) on the surface of CTL, which after binding to its receptor (Fas), triggers the apoptotic death of the Fas-expressing target cells. Previous experiments from our laboratory have shown that target-cell engagement leads to two “waves” of FasL surface expression on CTL. The first is thought to result from the rapid translocation of stored molecules and the second is believed to be product of the surface transport of newly synthesized proteins. The research objective for this work was to identify the storage compartment that harbors FasL in unstimulated CTL and to determine the trafficking route that the pre-synthesized stored pool of FasL molecules follow to reach their storage compartment. Using confocal microscopy colocalization analysis, I demonstrated that FasL is stored in intracellular vesicles that also contain the proteins Syntaxin 3, Munc18-2 and Rab32 and that these compartments are distinct from the lysosomal granules. Moreover, I found that FasL is endocytosed from the plasma membrane using a signal in its cytoplasmic tail and from there targeted to its storage vesicle via a tri-lysine motif. Furthermore, I found evidence that the motor protein myosin and the SNARE protein Syntaxin 3 affect the translocation of FasL to the surface. The findings presented in this report strongly indicated that the storage vesicle of FasL is distinct from lysosomal granules and suggested that it must thus be differentially regulated. It also provided markers for the FasL storage vesicle, and insight into the trafficking mechanism of this protein. Understanding how FasL trafficking is regulated will allow the manipulation of this killing pathway and to decipher its contribution during an immune response.