The membrane phospholipid phosphatidylinositol 4, 5-bisphosphate [PI (4,5)P2] is a critical signal transducer in eukaryotic cells. However, the physiological roles of the type I phosphatidylinositol phosphate kinases(PIPKIs) that synthesize PI(4,5) P2 are unknown. Here we show that the alpha isozyme of PIPKI (PIPKIalpha) negatively regulates mast cell functions and anaphylactic responses. In vitro, PIPKIalpha-deficient mast cells exhibit increased degranulation and cytokine production after Fc(epsilon)RI crosslinking. In vivo, PIPKIalpha-/- mice display enhanced passive cutaneous and systemic anaphylaxis. Mechanistically, filamentous actin was diminished in PIPKIalpha-/- mast cells and enhanced degranulation observed in the absence of PIPKIa was phenocopied in wild type mast cells treated with latrunculin, a pharmacological inhibitor of actin polymerization. Moreover, the association of Fc(epsilon)RI with lipid rafts and Fc(epsilon)RI mediated activation of signaling proteins is augmented in PIPKIalpha-/- mast cells. PIPKIalpha is thus a negative regulator of Fc(epsilon)RI-mediated cellular responses and anaphylaxis which functions by controlling the actin cytoskeleton and dynamics of Fc(epsilon)RI signaling. Our results are thus the first genetic evidence that PIPKI isoforms, and the pool of PI (4,5) P2 produced by each isoform, may be functionally specialized.