Exosomes are secreted vesicles formed in late endocytic compartments. Immature dendritic cells (DCs) secrete exosomes which transfer functional MHC-peptide complexes to other DCs. Since immature and mature DCs induce different functional T cell responses (i.e., tolerance versus priming), we asked whether DC maturation also influenced the priming abilities of their exosomes. We show that immature and mature murine DCs secrete morphologically similar exosomes. Extensive proteomic analysis of the two exosome populations showed identical overall protein composition, and provided an exhaustive image of the protein composition of DC-derived exosomes. By quantitative analysis, however, exosomes from mature DCs proved enriched in MHC class II, B7.2, ICAM-1, and depleted in MFG-E8, as compared to immature exosomes. In functional T cell stimulation assays, exosomes secreted by mature DCs were 50- to 100-fold more potent than exosomes from immature DCs, both in vitro and in vivo. MHC class II and ICAM-1 were necessary for the increased immune activity of exosomes secreted by mature DCs. Therefore, changes in protein composition and priming abilities of exosomes reflect the maturation signals received by DCs.