Multivesicular bodies (MVBs) are endocytic compartments that contain intraluminal vesicles formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these vesicles contain pro-apoptotic Fas ligand (FasL), which may be secreted as 'lethal exosomes' upon fusion of MVBs with the plasma membrane. Diacylglycerol kinase α (DGKα) regulate the secretion of exosomes, but it is unclear how this control is mediated. T-lymphocyte activation increases the number of MVBs that contain FasL. DGKα is recruited to MVBs and to exosomes in which it has a double function. DGKα kinase activity exerts a negative role in the formation of mature MVBs, as we demonstrate by the use of an inhibitor. Downmodulation of DGKα protein resulted in inhibition of both the polarisation of MVBs towards immune synapse and exosome secretion. The subcellular location of DGKα together with its complex role in the formation and polarised traffic of MVBs support the notion that DGKα is a key regulator of the polarised secretion of exosomes.