It has been suggested that mast cells might serve, under certain circumstances, as antigen-presenting cells (APCs) for T cells. However, whether cognate interactions between mast cells and class II–restricted CD4⁺ T cells actually occur is still an open question. We addressed this question by using peritoneal cell–derived mast cells (PCMCs) and freshly isolated peritoneal mast cells as APC models. Our results show that in vitro treatment of PCMCs with interferon-γ and interleukin-4 induced surface expression of mature major histocompatibility complex class II molecules and CD86. When interferon-γ/interleukin-4–primed PCMCs were used as APCs for CD4⁺ T cells, they induced activation of effector T cells but not of their naive counterparts as evidenced by CD69 up-regulation, proliferation, and cytokine production. Confocal laser scanning microscopy showed that CD4⁺ T cells formed immunological synapses and polarized their secretory machinery toward both antigen-loaded PCMCs and freshly isolated peritoneal mast cells. Finally, on cognate interaction with CD4⁺ T cells, mast cells lowered their threshold of activation via FcϵRI. Our results show that mast cells can establish cognate interactions with class II–restricted helper T cells, implying that they can actually serve as resident APCs in inflamed tissues.