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ATP is an important intercellular messenger in the extracellular space. In mast cells (MCs), ATP stimulates the ionotropic P2X4 receptor (P2X4R), resulting in enhanced degranulation and exacerbation of acute allergic reactions. In this study, we investigate whether ATP regulates inflammatory cytokine production in MCs. Gene expression was analyzed by quantitative RT-PCR, and cytokine production was measured using ELISA. The stimulation of mouse bone-marrow-derived MCs (BMMCs) with ATP alone had little effect on cytokine secretion. However, the co-stimulation with prostaglandin (PG) E2 resulted in a marked increase in the secretion of various cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and IL-13, accompanied by an increase in their mRNA levels. The effects of ATP were inhibited by P2X4R antagonists and diminished in BMMCs derived from P2X4R-deficient mice, suggesting that P2X4R mediated the reaction. The effects of PGE2 were mimicked by an EP3 receptor (EP3R) agonist and blocked by an EP3R antagonist. The synergistic cytokine mRNA elevations induced by ATP and PGE2 were blocked by nuclear factor-κB and Ca2+-calcineurin signaling inhibitors. Altogether, these results suggest that combining P2X4R and EP3R signaling enhances acute degranulation and the subsequent cytokine secretion, exacerbating allergic inflammation.