To investigate the signaling function of the Src‐family protein tyrosine kinase Lck in mature T cells, we generated transgenic mice that expressed Lck in thymocytes but not in peripheral lymphocytes. We compared the phenotype and signaling capacity of Lck‐deficient T cells with T cells from mice expressing a dominant inhibitory form of Lck and found that both mouse strains have diminished numbers of mature CD8⁺ T cells and respond poorly to CD28 costimulation. However, while T cells that lack Lck fail to mobilize Ca²⁺ after stimulation, those expressing the dominant negative protein do so normally. Our data demonstrate that Lck plays several unique roles in mature lymphocyte signaling.