Pre-TCR/CD3 signals are essential for survival and maturation of (CD44(-)25(+)) DN3 thymocytes via the (CD44(-)25(-)) DN4 stage to CD4(+)CD8(+) (DP) cells, a process termed beta-selection. The exact developmental stages of apoptosis resulting from lack of pre-TCR/CD3 signals have so far not been determined. Here we analyzed apoptotic cell death in relation to expression of clonotypic TCR polypeptides and to cell cycle status in immature thymocyte subpopulations of wild type (wt) mice and of several strains of mice with compromised pre-TCR/CD3 signaling complexes. In wt mice or pre-TCR/CD3-deficient mice, apoptotic cells could not be detected among DN3 cells but accumulated in a subset of DN4 expressing CD69. Apoptotic CD69(+)DN4 cells were rare in wt mice and were found among DN4 cells that were negative or low for intracellular TCRbeta and negative for TCRgamma delta polypeptide chains. Apoptotic CD69(+)DN4 cells were abundant in pre-TCR/CD3 signaling-deficient mice in which most DN4 cells failed to express clonotypic TCR polypeptides. Survival of DN4 cells, but not maturation of DN3 cells to DN4, was found to depend on the expression of clonotypic TCR polypeptides in the same cell. The results suggest that thymocytes unsuccessful in alpha beta or in gamma delta lineage development die by apoptosis in the DN4 subset.