ITAM-containing adaptors such as CD3ζ, FcεRIγ, and DAP12 are linked to a variety of activating receptors on NK cells and are thus presumed to be important for NK cell cytotoxicity, but whether these ITAM adaptors are unique or functionally redundant in the development of cytotoxicity has remained elusive. Two receptors expressed on NK cells, 2B4 and NKp46, can activate "natural cytotoxicity" against EBV transformed B cells or redirect ADCC. We therefore examined the requirement for ITAM adaptors in 2B4 and NKp46-initiated cytotoxicity using siRNA suppression. Here we provide evidence that FcεRIγ is required for 2B4-initiated cytotoxicity in redirected ADCC assays, whereas CD3ζ and DAP12 are not. Importantly, 2B4 surface levels were not altered by suppression of FcεRIγ, suggesting the possibility that this ITAM adaptor was functioning to costimulate 2B4-initiated killing. Interestingly, FcεRIγ, but not CD3ζ, was important for NKp46-initated cytotoxicity in redirected ADCC assays and siRNA toward FcεRIγ led to a significant reduction in NKp46 surface levels. FcεRIγ is thus an important ITAM adaptor molecule for cytotoxicity initiated by 2B4 through the regulation of NKp46 cell surface levels. FcεRIγ may also be important for killing EBV transformed cells since 2B4 and NKp46 are involved in killing EBV transformed B cells. This work was supported by NCI grant CA47752 to D.D.B. and NIH grant F31-AI75632 to A.T.B.