<p>T cell receptor (TCR) signaling is critically dependent on the Src-family kinase Lck, whose activation is tightly regulated both spatially and conformationally during antigen recognition. Here, we employ an improved second-generation FRET-based biosensor (TqLckV2.3) to visualize Lck conformational dynamics in live T cells with high spatial resolution. Upon TCR engagement, we observe a paradoxical increase in whole-cell FRET signal, which immunolabeling reveals to be due to selective internalization of inactive Lck (pY⁵⁰⁵), while active Lck (pY³⁹⁴) remains membrane-associated and enriched at the immune synapse (IS). Using CD8α mutants that disrupt Lck binding, we demonstrate that free Lck undergoes more pronounced conformational activation than CD8-bound Lck. Furthermore, we show that C-terminal Src kinase (Csk) preferentially phosphorylates free Lck at Y⁵⁰⁵, while CD45 suppresses its activation via dephosphorylation of Y³⁹⁴, suggesting a dual regulatory mechanism that maintains free Lck in an inactive state under resting conditions. High-resolution imaging confirms sustained activation of Lck at the IS and transient inactivation at the periphery, revealing a spatially confined signaling architecture. These findings uncover a novel regulatory mechanism involving selective internalization and spatial segregation of Lck conformations and establish TqLckV2.3 as a powerful tool for dissecting TCR signaling dynamics.</p>