Exosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. The mechanisms involved in their biogenesis remain so far unclear although they constitute targets to modulate exosome formation and therefore are a promising tool to understand their functions. We have performed an RNA interference screen targeting twenty-three components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins in MHC class II (MHC II)-expressing HeLa-CIITA cells. Silencing of HRS, STAM1, or TSG101 reduced the secretion of EV-associated CD63 and MHC II but each gene altered differently the size and/or protein composition of secreted EV, as quantified by immuno-electron microscopy. By contrast, depletion of VPS4B augmented this secretion while not altering the features of EVs. For several other ESCRT subunits, the screen did not allow to conclude on their involvement in exosome biogenesis. Interestingly, silencing of ALIX increased MHC II exosomal secretion, due to an overall increase in intracellular MHC II protein and mRNA levels. In human dendritic cells (DCs), ALIX depletion also increased MHC II in the cells, but not in the released CD63-positive EVs. Such differences could be attributed to a higher heterogeneity in size, and higher MHC II and lower CD63 contents in vesicles recovered from DCs as compared to HeLa-CIITA. The results reveal a role for selected ESCRT components and accessory proteins in exosome secretion and composition by HeLa-CIITA. They also highlight biogenetic differences in vesicles secreted by a tumour cell line and primary DCs.