Previous studies have shown that pharmacological inhibition of the phosphoinositol-3 (PI3) kinase disrupts the activation of mast cells. Through phosphoinositide-dependent kinase PDK1, PI3 kinase activates the serum- and glucocorticoid-inducible kinase 3 (SGK3). The present study explored the role of SGK3 in mast cell function. Mast cells were isolated and cultured from bone marrow (BMMCs) of gene-targeted mice lacking SGK3 ( sgk3 −/−) and their wild-type littermates ( sgk3 +/+). BMMC numbers in the ear conch were similar in both genotypes. Stimulation with IgE and cognate antigen triggered the release of intracellular Ca2+ and entry of extracellular Ca2+. Influx of extracellular Ca2+ but not Ca2+ release from intracellular stores was significantly blunted in sgk3 −/− BMMCs compared with sgk3 +/+ BMMCs. Antigen stimulation further led to a rapid increase of a K+-selective conductance in sgk3 +/+ BMMCs, an effect again blunted in sgk3 −/− BMMCs. In contrast, the Ca2+ ionophore ionomycin activated K+ currents to a similar extent in sgk3 −/− and in sgk3 +/+ BMMCs. β-Hexosaminidase release, triggered by antigen stimulation, was also significantly decreased in sgk3 −/− BMMCs. IgE-dependent anaphylaxis measured as a sharp decrease in body temperature upon injection of DNP-HSA antigen was again significantly blunted in sgk3 −/− compared with sgk3 +/+ mice. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk3 −/− than in sgk3 +/+ mice. In conclusion, both in vitro and in vivo function of BMMCs are impaired in gene targeted mice lacking SGK3. Thus SGK3 is critical for proper mast cell function.