Exosomes are 60–100-nm membrane vesicles secreted by cells, including B cells, and are formed by the fusion of multivesicular bodies (MVB) with the plasma membrane. This study characterized the protein and lipid composition of highly purified human B cell-derived exosomes. Mass spectrometry revealed the presence of MHC class I and II, heat shock proteins, integrins, and tetraspanins, among other proteins. Exosomes were enriched in cholesterol, sphingomyelin, and ganglioside GM3, which are typically found in detergent-resistant membranes (DRMs). Most exosome-associated proteins, including MHC class II and tetraspanins, were insoluble in CHAPS-containing buffers, suggesting their incorporation into tetraspanin-containing DRMs. These findings indicate that proteins are sorted into MVB internal vesicles through their incorporation into tetraspanin-containing DRMs. MVBs are endosomes that accumulate vesicles in their lumen, leading to their transformation into MVBs. Proteins sorted to MVB internal vesicles can have three fates: degradation, temporary storage, or release as exosomes. Exosomes derived from B cells carry MHC class II and can stimulate CD4+ T cells and bind to follicular dendritic cells. Exosomes from dendritic cells loaded with tumor-derived peptides can stimulate cytotoxic T lymphocytes. Exosomes are released by various cell types, including B cells, and may serve multiple functions in intercellular communication. Exosomes contain proteins and lipids that are characteristic of MVBs, including those involved in protein sorting and MVB formation. The presence of tetraspanins and DRM-like properties in exosomes suggests that they may contribute to protein sorting in MVBs and membrane budding. The study also found that exosomes are enriched in cholesterol, sphingomyelin, and GM3, which are characteristic of DRMs. These findings support the idea that exosomes contain protein-lipid complexes that can be described as DRMs or raft-like structures. The presence of MHC class II in exosomes and MVBs, but not at the plasma membrane, suggests that MHC class II is associated with DRMs in these structures. The study also identified the presence of Gα2 in exosomes, which may be targeted to MVB internal vesicles due to its incorporation into lipid rafts at the MVB-limiting membrane. The presence of hsc70 in exosomes is consistent with its detection in other cell types, and its role in protein sorting and folding is supported by its presence in exosomes. The study also identified the presence of clathrin in exosomes, which may be involved in MVB formation and protein sorting. Overall, the study provides insights into the composition and function of exosomes, highlighting their potential role in intercellular communication and immune responses.Exosomes are 60–100-nm membrane vesicles secreted by cells, including B cells, and are formed by the fusion of multivesicular bodies (MVB) with the plasma membrane. This study characterized the protein and lipid composition of highly purified human B cell-derived exosomes. Mass spectrometry revealed the presence of MHC class I and II, heat shock proteins, integrins, and tetraspanins, among other proteins. Exosomes were enriched in cholesterol, sphingomyelin, and ganglioside GM3, which are typically found in detergent-resistant membranes (DRMs). Most exosome-associated proteins, including MHC class II and tetraspanins, were insoluble in CHAPS-containing buffers, suggesting their incorporation into tetraspanin-containing DRMs. These findings indicate that proteins are sorted into MVB internal vesicles through their incorporation into tetraspanin-containing DRMs. MVBs are endosomes that accumulate vesicles in their lumen, leading to their transformation into MVBs. Proteins sorted to MVB internal vesicles can have three fates: degradation, temporary storage, or release as exosomes. Exosomes derived from B cells carry MHC class II and can stimulate CD4+ T cells and bind to follicular dendritic cells. Exosomes from dendritic cells loaded with tumor-derived peptides can stimulate cytotoxic T lymphocytes. Exosomes are released by various cell types, including B cells, and may serve multiple functions in intercellular communication. Exosomes contain proteins and lipids that are characteristic of MVBs, including those involved in protein sorting and MVB formation. The presence of tetraspanins and DRM-like properties in exosomes suggests that they may contribute to protein sorting in MVBs and membrane budding. The study also found that exosomes are enriched in cholesterol, sphingomyelin, and GM3, which are characteristic of DRMs. These findings support the idea that exosomes contain protein-lipid complexes that can be described as DRMs or raft-like structures. The presence of MHC class II in exosomes and MVBs, but not at the plasma membrane, suggests that MHC class II is associated with DRMs in these structures. The study also identified the presence of Gα2 in exosomes, which may be targeted to MVB internal vesicles due to its incorporation into lipid rafts at the MVB-limiting membrane. The presence of hsc70 in exosomes is consistent with its detection in other cell types, and its role in protein sorting and folding is supported by its presence in exosomes. The study also identified the presence of clathrin in exosomes, which may be involved in MVB formation and protein sorting. Overall, the study provides insights into the composition and function of exosomes, highlighting their potential role in intercellular communication and immune responses.