Mast cells play a pivotal role in innate host immune response to gram-negative bacteria. We report that Janus kinase 3 plays a role in mast cell-mediated bacterial clearance and neutrophil recruitment by regulating the release of tumor necrosis factor from mast cells. The role of JAK3 in mast cell-facilitated neutrophil recruitment and bacterial clearance was investigated by comparing the neutrophil influxes and bacterial clearance in mast cell-deficient W/W(v) mice reconstituted with JAK3(+/+) or JAK(-/-) mast cells. The neutrophil influx, bacterial clearance, and survival outcome in W/W(v) mice reconstituted with JAK3(+/+) mast cells was better than in W/W(v) mice reconstituted with JAK3(-/-) mast cells. These findings provide evidence that JAK3 is a key regulator of mast cell-mediated innate immunity against gram-negative bacteria.