Engagement of the T cell antigen receptor (TCR) induces the transphosphorylation of the zeta chain-associated protein of 70,000 Mr (ZAP-70) protein tyrosine kinase (PTK) by the CD4/8 coreceptor associated Lck PTK. Phosphorylation of Tyr 493 within ZAP-70's activation loop results in the enzymatic activation of ZAP-70. Additional tyrosines (Tyrs) within ZAP-70 are phosphorylated that play both positive and negative regulatory roles in TCR function. Phosphorylation of Tyr residues (Tyrs 315 and 319) within the Interdomain B region of the ZAP-70 PTK plays important roles in the generation of second messengers after TCR engagement. Here, we demonstrate that phosphorylation of these two Tyr residues also play important roles in mediating the positive and negative selection of T cells in the thymus.