Molecular adaptors/scaffolds have indispensable roles in the activation of lymphocytes. In this report, we have demonstrated the role of tyrosine phosphorylation of an adaptor protein 3BP2 (c-Abl-SH3 domain binding protein-2, also known as SH3BP2) in T cell receptor (TCR)-mediated activation of transcription factor. Short interfering RNA for 3BP2 suppresses the expression level of endogenous 3BP2 and inhibits TCR-mediated activation of interleukin (IL)-2 promoter and nuclear factor of activated T cells (NFAT) element. Engagement of TCR induces tyrosine phosphorylation and lipid raft translocation of 3BP2. The overexpression studies reveal that substitution of 3BP2-Tyr¹⁸³, Tyr⁴⁴⁶, or Arg⁴⁸⁶ in the SH2 domain suppresses TCR-mediated activation of NFAT. Point mutations of 3BP2 cannot affect the translocation of 3BP2 into the lipid raft. Phosphorylation of Tyr¹⁸³ is required for the interaction with Vav1, the guanine nucleotide exchanging factor of Rac1. In fact, overexpression of dominant-negative form of Rac1 inhibits TCR-mediated activation of NFAT. Phosphorylation of Tyr⁴⁴⁶ recruits the SH2 domain of Lck for the optimal activation of transcription factors. Furthermore, point mutation of Arg⁴⁸⁶ in the 3BP2-SH2 domain that couples ZAP-70 to LAT dramatically reduces NFAT activation. These results suggest that the site-directed functions of 3BP2 induce the activation of transcription factors.