Exosomes are small vesicles (60–100 nm) secreted by various cell types upon the fusion of endosomal compartments with the plasma membrane. Exosomes from antigen‐presenting cells (APC), such as B lymphocytes and dendritic cells (DC), bear MHC class II molecules. In addition, the injection of DC‐derived exosomes was reported to elicit potent T cell responses in vivo. Here, we analyzed the activation of specific T cells by MHC class II‐bearing exosomes in vitro. The rat mast cell line, RBL‐2H3, was engineered to express human class II molecules uniformly loaded with an antigenic peptide [HLA‐DR1–hemagglutinin (HA)]. These cells secreted exosomes bearing DR1 class II molecules upon stimulation by a calcium ionophore or IgE receptor cross‐linking. Exosomes bearing DR1–HA(306–318) complexes activated HA/DR1‐specific T cells only weakly, whereas the cross‐linking of such exosomes to latex beads increased stimulation of specific T cells. By contrast, the incubation of free exosomes with DC resulted in the highly efficient stimulation of specific T cells. Thus, exosomes bearing MHC class II complexes must be taken up by professional APC for efficient T cell activation.