Exosomes are secreted vesicles that are formed during endosome maturation in multivesicular bodies (MVBs). Some MVBs are degraded but other can fuse with the plasma membrane and, thus, secrete their internal vesicles as exosomes. Despite extensive research efforts, the exact mechanisms that underly exosome biogenesis and cargo sorting within these vesicles remain poorly understood, although components of the endosomal sorting complex required for transport (ESCRT) have been implicated in their function. In this work (p. 5553), Clotilde Théry, Graça Raposo and colleagues use RNA interference (RNAi) to target 23 different components of the four complexes (ESCRT-0, -I, II and III) as well as associated proteins in major histocompatibility complex class II (MHC II)-expressing HeLa cells, which allow the monitoring of exosome secretion. They identify five ESCRT components that appear to be involved in exosome biogenesis or secretion; knockdown of three ESCRT-0 and/or ESCRT-I factors decreases exosome secretion, whereas silencing of two accessory proteins either increases secretion or modifies the MHC II content. Interestingly, by using immuno-electron microscopy, the authors show that silencing of these factors also differentially affects size and protein composition of the secreted vesicles, suggesting that exosomes exhibit a considerable heterogeneity. Thus, the authors propose that ESCRTs have a role in the formation of exosomes from a subpopulation of vesicles within MVBs as well as in their secretion.