The enzymatic activity of the Src family tyrosine kinase p56^Lck (Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr³⁹⁴ and Tyr⁵⁰⁵. Phosphorylation of Tyr³⁹⁴ and the conformational opening of Lck are believed to activate the kinase, whereas Tyr⁵⁰⁵ phosphorylation is thought to generate a closed, inactive conformation of Lck. We investigated whether the conformation of Lck and its phosphorylation state act in concert to regulate the initiation of T cell receptor (TCR) signaling. With a sensitive biosensor, we used fluorescence lifetime imaging microscopy (FLIM) to investigate the conformations of wild-type Lck and its phosphorylation-deficient mutants Y394F and Y505F and the double mutant Y394F/Y505F in unstimulated T cells and after TCR stimulation. With this approach, we separated the conformational changes of Lck from the phosphorylation state of its regulatory tyrosines. We showed that the conformational opening of Lck alone was insufficient to initiate signaling events in T cells. Rather, Lck additionally required phosphorylation of Tyr³⁹⁴ to induce T cell activation. Consistent with the FLIM measurements, an optimized immunofluorescence microscopy protocol revealed that the TCR-stimulated phosphorylation of Lck at Tyr³⁹⁴ occurred preferentially at the plasma membrane of Jurkat cells and primary human T cells. Our study supports the hypothesis that T cell activation through the TCR complex is accompanied by the de novo activation of Lck and that phosphorylation of Tyr³⁹⁴ plays a role in Lck function that goes beyond inducing an open conformation of the kinase.