Exosomes from APC are nano-vesicles that can induce antigen-specific T cell responses and are presently explored as therapeutic tools in different clinical settings. Investigations of the capacity of exosomes to stimulate T cells in vitro have mostly been performed on T cell hybridomas, clones or lines. Whether exosomes can stimulate T cells directly or need the presence of dendritic cells (DC) is debated. We could detect exosome-induced antigen-specific CD8(+) T cell responses in peripheral blood from humans. Exosomes from monocyte-derived DC (MDDC) were loaded with a mix of 23 immunogenic peptides from EBV, CMV and influenza virus, and added to autologous peripheral CD8(+) T cells. IFN-gamma-producing cells were detected by enzyme-linked immunospot assay (ELISPOT). MDDC-exosomes induced IFN-gamma production in CD8(+) T cells without addition of DC. The response was exosome dose dependent, and dependent on exosomal MHC class I. Furthermore, we detected an enhanced T cell stimulatory capacity by exosomes from lipopolysaccharide-matured MDDC compared to exosomes from immature MDDC. Exosomes could also induce TNF-alpha production. These results show, for the first time, that exosomes can directly stimulate human peripheral CD8(+) T cells in an antigen-specific manner and that ELISPOT is a suitable method for detecting exosome-induced peripheral T cell responses. This system may provide a useful tool when developing exosomes as therapeutic agents.