Activated mast cells are a major source of the eicosanoids PGD 2 and leukotriene C 4 (LTC 4), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A 2, thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC 4 and PGD 2, respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcεRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcεRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcεRI-dependent PGD 2, LTC 4, and ROS production in bone marrow-derived mast cells of Btk−/− mice. The FcεRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A 2 phosphorylation but rather appears to regulate the generation of ROS, LTC 4, and PGD 2 by contributing to the necessary Ca 2+ signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2-and PI3K/Btk-dependent pathways.