Mast cells play an important role in host defense against various pathogens, but their role in viral infection has not been clarified in detail. dsRNA, synthesized by various types of viruses and mimicked by polyinosinic-polycytidylic acid (poly(I:C)) is recognized by Toll-like receptor 3 (TLR3). In this study, we demonstrate that poly(I:C) injection in vivo potently stimulates peritoneal mast cells to up-regulate a number of different costimulatory molecules. Therefore, we examined the expression and the functional significance of TLR3 activation in mast cells. Mast cells express TLR3 on the cell surface and intracellularly. After stimulation of mast cells with poly(I:C) and Newcastle disease virus (NDV), TLR3 is phosphorylated and the expression of key antiviral response cytokines (interferon β, ISG15) and chemokines (IP10, RANTES) is upregulated. Interestingly, mast cells activated via TLR3-poly(I:C) potently stimulate CD8⁺ T-cell recruitment. Indeed, mast-cell–deficient mice (Kit^W/Kit^W-v) given an intraperitoneal injection of poly(I:C) show a decreased CD8⁺ T-cell recruitment, whereas granulocytes normally migrate to the peritoneal cavity. Mast-cell reconstitution of Kit^W/Kit^W-v mice normalizes the CD8⁺ T-cell influx. Thus, mast cells stimulated through engagement of TLR3 are potent regulators of CD8⁺ T-cell activities in vitro and in vivo.