Costimulatory signals play an important role in the development of T helper cell type 1 (Th1) or Th2 type. Little is known about jacalin plus CD28-mediated signaling and cytokine secretion. In the present study, we analyzed the intracellular signaling events following stimulation of CD4+ T cells with jacalin plus CD28 cross-linking (CD28XL) with anti-CD28 antibody. Our results indicate enhanced phosphorylation of Tec and linker for activation of T cells when compared with stimulation with jacalin alone or CD28XL alone. Stimulation with jacalin or CD28XL appears to be insufficient to induce interleukin (IL)-4 secretion; however, CD28XL followed by stimulation with jacalin resulted in enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and increased secretion of IL-4. However, compared with stimulation with phorbol 12-myristate 13-acetate plus ionomycin, jacalin plus CD28XL resulted in decreased levels of tumor necrosis factor alpha secretion. Addition of p38 inhibitor, SB203580, inhibited p38 phosphorylation and IL-4 secretion. These data suggest that jacalin stimulation alone appears to be insufficient for Th2 development, and addition of CD28 costimulation induced Th2 generation. We propose that jacalin plus CD28XL induces Th2 differentiation via activation of p38 MAPK.