Interaction of the CD4 co-receptor with major histocompatibility complex (MHC) class II molecules during antigen presentation results in enhancement of antigen receptor signaling. The synergism between the two receptors is believed to result from the juxtaposition of the CD4-associated tyrosine kinase p56lck with the cytoplasmic domains of CD3 complex components. Here, we report that cross-linking of CD4 on the surface of Jurkat cells using monoclonal antibodies results in activation of the CD3-associated kinase p59fyn. Co-cross-linking of CD4 and CD3 results in synergistic activation of p59fyn. The p59fyn kinase is also hyperactive in a Jurkat cell line stably transfected with a constitutively active p56lck mutant, indicating that p56lck mediates CD4 activation of p59fyn. In support of this hypothesis, expression of a dominant inhibitory mutant of p59fyn blocks CD4 signals involved in gene activation. In addition, the p59fyn dominant inhibitor mutant blocks gene-activating signals induced by expression of a constitutively active mutant of p56lck. Overexpression of the regulatory kinase p50csk, which attenuates TcR signaling by inactivation of p59fyn, inhibits signaling from the constitutively active form of p56lck. Taken together, these data suggest that CD4/p56lck enhancement of TcR signaling is, at least in part, mediated by activation of p59fyn, and may be regulated by p50csk.