The characteristics, importance, and molecular requirements for interactions between mast cells (MCs) and CD8+ T cells have not been elucidated. Here, we demonstrated that MCs induced antigen-specific CD8+ T cell activation and proliferation. This process required direct cell contact and MHC class I-dependent antigen cross-presentation by MCs and induced the secretion of interleukin-2, interferon-γ, and macrophage inflammatory protein-1α by CD8+ T cells. MCs regulated antigen-specific CD8+ T cell cytotoxicity by increasing granzyme B expression and by promoting CD8+ T cell degranulation. Because MCs also upregulated their expression of costimulatory molecules (4-1BB) and released osteopontin upon direct T cell contact, MC-T cell interactions probably are bidirectional. In vivo, adoptive transfer of antigen-pulsed MCs induced MHC class I-dependent, antigen-specific CD8+ T cell proliferation, and MCs regulated CD8+ T cell-specific priming in experimental autoimmune encephalomyelitis. Thus, MCs are important players in antigen-specific regulation of CD8+ T cells. © 2009 Elsevier Inc. All rights reserved.