CD63 is a tetraspanin, which organize on cholesterol enriched microdomains, especially on late endosomes. Previous work of our team has shown that CD63 could regulate the sorting of cargoes into ILVs through an ESCRT-independent manner in melanocytes. However, CD63 has also been involved in other trafficking events in other cell type and so far, we have no clear idea of the general process that CD63 would control and would unify these disparate reports. In order to elucidate to general role of CD63 we compared two cell lines, Hela and MNT-1 that we Knock-out for CD63 by CRISPR in both cell lines and investigated the EV generation and the expression level of proteins involved in this process in cell lysates and the EV pellet obtained by SEC.