Eph kinases are the largest family of receptor tyrosine kinases (RTK), and their ligands are cell surface molecules. The known functions of Eph kinases are mainly pattern formation in the CNS. Although several Eph kinases are expressed at high levels in hemopoietic cells and in the thymus, we have no knowledge of the functions of any Eph kinase in the immune system. In this study, we have demonstrated that an Eph kinase, EphB6, was expressed at high levels in Jurkat leukemic T cells. Co-cross-linking of EphB6 and CD3 led to an altered profile of lymphokine secretion along with proliferation inhibition of Jurkat cells. The cells subsequently underwent Fas-mediated apoptosis. Although EphB6 has no intrinsic kinase activity, its cross-linking triggered general protein tyrosine phosphorylation in Jurkat cells. EphB6 was found to associate with a number of molecules in the signaling pathways, notably Cbl. EphB6 cross-linking resulted in Cbl dephosphorylation and dissociation from Src homology 2 domain-containing tyrosine phosphatase-1 (SHP-1). Our results show that EphB6 has important functions in T cells, and it can transduce signals into the cells via proteins it associates with.