Although there was no impairment in IL-2 secretion and proliferation of Fyn-deficient naïve CD4 cells after stimulation with antigen and antigen-presenting cells, stimulation of these cells with anti-CD3 and anti-CD28 revealed profound defects. Crosslinking of purified wild-type naïve CD4 cells with anti-CD3 activated Lck and initiated the signaling cascade downstream of Lck, including phosphorylation of ZAP-70, LAT, and PLC-γ1; calcium flux; and dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT)p. All of these signaling events were diminished severely in Fyn-deficient naïve cells activated by CD3 crosslinking. Coaggregation of CD3 and CD4 reconstituted this Lck-dependent signaling pathway in Fyn^-/- T cells. These results suggest that when signaling of naïve T cells is restricted to the T cell antigen receptor, Fyn plays an essential role by positive regulation of Lck activity.