Mast cells (MCs) produce a variety of chemokines and cytokines to induce allergic inflammation. Previously, we have shown that co-stimulation with ATP and prostaglandin (PG) E₂ synergistically increases the secretion of various inflammatory cytokines via P2X4 and EP₃ receptors, respectively. In the present study, we examined the mechanism underlying the synergistic cytokine production by co-stimulation of P2X4 and EP₃ receptors in mouse bone marrow-derived MCs (BMMCs). Stimulation of BMMCs with PGE₂ elicited rapid phosphorylation of ERK1/2, p38 MAP kinase, and Akt. In contrast, ATP alone had only weak effects on phosphorylation of these signaling molecules, and little affected PGE₂-induced responses. Although ATP and PGE₂ hardly induced NF-kB p65 phosphorylation, co-stimulation with them elicited an increase in NF-kB p65 phosphorylation. This effect was absent in BMMCs obtained from P2X4 receptor deficient mice. The cytokine production and NF-kB p65 phosphorylation induced by co-stimulation with ATP and PGE₂ were suppressed by inhibitors of NF-kB and phosphatidylinositol-3-kinase (PI3K). These results suggest that co-stimulation of P2X4 and EP₃ receptors enhances MC cytokine production by activating NF-kB signaling pathway in a PI3K-dependent manner