Activation through the T cell receptor (TCR) initiates a signaling cascade in T cells that induces extensive molecular and cellular changes. The adaptor protein Linker for Activation of T cells (LAT) plays an essential role in transducing activation and regulatory signals downstream of the TCR. Phosphorylation of LAT tyrosine residues recruits multiple signaling proteins, leading to the assembly of the LAT signalosome, which is crucial for relaying signals that regulate T cell development and function. We previously showed that substitution of a negatively charged amino acid segment preceding the fifth tyrosine residue of LAT (Tyr127 in humans or Tyr132 in mouse LAT) enhances some early TCR signaling events, whereas downstream responses, such as Ca2+ influx and Erk phosphorylation, are partially inhibited. To investigate the physiological relevance of this segment in vivo, we generated a new LAT knock-in mouse strain (LatNIL) in which the negatively charged segment was replaced with a non-charged sequence. Unexpectedly, this mutation led to an alternative splicing event in the Lat gene that excluded exons 6 and 7, resulting in a frameshift, a premature stop codon at residue 145, and the loss of the six C-terminal tyrosine residues of LAT. Homozygous LatNIL/NIL mice showed a phenotype similar to that of LAT-knockout and Lat4YF mice (in which the four C-terminal tyrosines had been mutated to phenylalanine). Interestingly, homozygous LatNIL/NIL mice exhibited a distinct population of γδ T cells in lymphoid organs, which has not been observed in LAT-KO or Lat4YF mice. These γδ T cells expressed higher levels of CD27 compared to those in wild-type and LAT-KO mice, suggesting altered activation or differentiation states. Together, these data highlight how subtle alterations in LAT structure can profoundly impact T cell signaling and lineage composition.