Stage-specific expression of a number of cell-surface and signaling proteins is critical for normal development of T cells in the thymus. Equally important may be the loss of expression/signaling of developmentally regulated proteins for proper transitioning of developing T cells into thymic subsets. Ly-6A.2 exhibits a regulated pattern of expression on T cells maturing in the thymus, and dysregulating its expression results in arrest of developing T cells within the CD3−CD4−CD8− triple negative (TN) stage where the normal expression of Ly-6A.2 is extinguished. To further characterize the mechanisms underlying this block, we examined whether cell signaling and/or cell adhesion properties of the Ly-6A.2 molecule influenced the block in T-cell development. Analysis of bone marrow chimeras generated by injecting CFSE-labeled Ly-6A.2 transgenic bone marrow cells into irradiated syngeneic non-transgenic mice revealed normal trafficking of developing T cells from the cortex into the medulla. Production of LAT but not p56lck was diminished in CD4−CD8− DN cells from Ly-6A.2 dysregulated mice when compared with control littermates. Dysregulated expression of Ly-6A.2 did not suppress endogenous TCR-Vβ expression. Finally, dysregulated expression of Ly-6A.2 enhanced apoptosis of an immature CD4+CD8+ (DP) subset of developing cells and altered the selected TCR-Vβ repertoire. Taken together, these observations indicate that the termination of Ly-6A.2 expression and signaling within the CD4−CD8−CD3− subset of developing T cells is an important checkpoint during normal thymic development.