Stimulation of thymocytes or mature T cells via the T cell receptor (TcR)/CD3 complex activates a cascade of processes inducing cells to enter the cell cycle. A key step is the activation of phosphatidylinositol‐specific phospholipase C (PI‐PLC) within seconds following TcR/CD3 stimulation, an event which is strongly enhanced by co‐ligation of the CD4 (or CD8) accessory molecule with TcR/CD3. In contrast, co‐ligation of CD45 inhibits the same TcR/CD3 responses. The machinery which couples the TcR/CD3 complex, CD4, and CD45 to PI‐PLC appears to involve regulation of tyrosine phosphorylation, as the TcR/CD3 and CD4 receptors are associated with the tyrosine kinases p59^fyn and p56^lck, respectively, and CD45 has intrinsic tyrosine phosphatase activity. Here, we have examined the ability of CD45 to regulate signal transduction via TcR/CD3 in human thymocytes. Co‐cross‐linking CD45 to the TcR/CD3 complex strongly suppressed the tyrosine phosphorylation of several intracellular substrates normally seen following TcR/CD3 stimulation. This effect of CD45 was associated with inhibition of a rise in intracellular calcium following TcR/CD3 ligation. Since TcR/CD3 stimulation of mature T cells induces tyrosine phosphorylation of PLC_γ1, we investigated this phenomenon in thymocytes, and asked whether ligation of CD45 might regulate this process. By immunoprecipitation we found that TcR/CD3 stimulation induced tyrosine phosphorylation of PLC_γ1, an effect which was enhanced by co‐cross‐linking CD4 to TcR/CD3. In contrast, co‐ligation of CD45 strongly blocked PLC_γ1 phosphorylation induced by either stimulus. Consistent with previous findings in mature T cells, CD45 cross‐linking was able to partially inhibit TcR/CD3‐induced thymocyte proliferation when interleukin 2 was used as a second signal, but almost completely (80%–90%) blocked proliferation when anti‐CD28 mAb was used as the second signal, suggesting that CD45 cross‐linking may be able to block interleukin 2 production via the CD28 pathway. These effects of CD45 on TcR/CD3 signaling and proliferation in thymocytes point towards a potential role for this pathway in thymic selection.