Natural killer (NK) cells participate in both innate and adaptive immunity through the prompt secretion of cytokines and ability to lyse virally infected cells or tumor cells. Triggering of NK cells requires aggregation of surface receptors such as CD2 and CD16, and NK cell activity can be augmented in vitro by stimulation with IL-2. In this study, we examined the role of adapter proteins in the increased NK activation following CD2 crosslinking and IL-2 stimulation of NK3.3 cells. NK3.3 cells lysed NK-sensitive K562 cells in a CD2-dependent manner, and IL-2 markedly enhanced lytic activity in a 4h cytotoxic assay. IL-2 also enhanced spontaneous and CD2-mediated granule exocytosis from NK3.3 cells. CD2 crosslinking markedly induced tyrosine phosphorylation of Cbl associated with Grb2 or CrkL, Shc and LAT, compared with IL-2 stimulation. However, costimulation of IL-2 with CD2 crosslinking remarkably enhanced associations of Grb2-Shc and CrkL-Cbl, compared to IL-2 stimulation or CD2 crosslinking alone. In vitro binding studies using GST-fusion proteins revealed that interactions of Grb2-Shc and CrkL-Cbl were mediated through each SH2 domain in tyrosine phosphorylation-dependent manner. Furthermore, CD2 crosslinking, but not IL-2 stimulation, markedly induced tyrosine phosphorylation of LAT. Thus, tyrosine phosphorylation of different adapter proteins and consequent interactions between signaling molecules described here may explain the molecular mechanisms of the additive effects of IL-2 stimulation and CD2 crosslinking on NK cell activation.