Ligation of high-affinity IgE receptor I (FcεRI) on RBL-2H3 cells leads to recruitment of FcεRI and type II phosphatidylinositol 4-kinases (PtdIns 4-kinases) into lipid rafts. Lipid raft integrity is required for the activation of type II PtdIns 4-kinases and signal transduction through FcεRIγ during RBL-2H3 cell activation. However, the molecular mechanism by which PtdIns 4-kinases are coupled to FcεRI signaling is elusive. Here, we report association of type II PtdIns 4-kinase activity with FcεRIγ subunit in anti-FcεRIγ immunoprecipitates. FcεRIγ-associated PtdIns 4-kinase activity increases threefold upon FcεRI ligation in anti-FcεRIγ immunoprecipitates. Biochemical characterization of PtdIns 4-kinase activity associated with FcεRIγ reveals that it is a type II PtdIns 4-kinases. Canonical tyrosine residues mutation in FcεRIγ ITAM (Y65 and Y76) reveals that these two tyrosine residues in γ subunit are required for its interaction with type II PtdIns 4-kinases.