Thymic selection is a tightly regulated developmental process essential for establishing central tolerance. The intensity of TCR-mediated signaling is a key factor for determining cell fate in the thymus. It is widely accepted that low-intensity signals result in positive selection, whereas high-intensity signals induce negative selection. Transmembrane adaptor proteins have been demonstrated to be important regulators of T cell activation. However, little is known about their role during T cell development. Herein, we show that SIT (SHP2 Src homology domain containing tyrosine phosphatase 2-interacting transmembrane adaptor protein) and TRIM (TCR-interacting molecule), two structurally related transmembrane adaptors, cooperatively regulate TCR signaling potential, thereby influencing the outcome of thymic selection. Indeed, loss of both SIT and TRIM resulted in the up-regulation of CD5, CD69, and TCRbeta, strong MAPK activation, and, consequently, enhanced positive selection. Moreover, by crossing SIT/TRIM double-deficient mice onto transgenic mice bearing TCRs with different avidity/affinity, we found profound alterations in T cell development. Indeed, in female HY TCR transgenic mice, positive selection was completely converted into negative selection resulting in small thymi devoided of double-positive thymocytes. More strikingly, in a nonselecting background, SIT/TRIM double-deficient single-positive T cells developed, were functional, and populated the periphery. In summary, we demonstrated that SIT and TRIM regulate cell fate of developing thymocytes, thus identifying them as essential regulators of central tolerance.