Abstract CD226 is known to be expressed on many types of peripheral lymphoid cells and involved in T cell differentiation, activation, and cytotoxicity. In this study, we report that CD226 is also expressed on mouse thymocytes at varying developmental stages, and its expression is associated with resistance of thymocytes to apoptosis. The levels of CD226 expression appeared to be closely coupled with thymocyte development, in that it was preferentially expressed on CD4+CD8− and CD4−CD8+ thymocytes at all stages during mouse development, and was markedly increased on the cells in neonatal mice. Of the CD4+CD8+ population, CD226 was predominantly expressed by the cells also positive for CD69, suggesting that CD226 expression may be induced in thymocyte-positive selection. Inhibition of CD226 by short hairpin RNA in a fetal thymus organ culture model led to reduced thymus cellularity, which was associated with enhanced apoptotic cell death. In contrast, CD226-transgenic mice displayed enlarged thymus lobes resulting from increased thymus cellularity. CD226 on thymocytes seemed to play a role in regulating the expression of survivin, as inhibition of CD226 down-regulated survivin, but overexpression of CD226 rescued thymocytes from apoptosis through up-regulation of survivin. In addition, overexpression of CD226 reduced sensitivity of EL-4 thymoma cells to apoptosis by up-regulating the expression of survivin. Taken together, these results indicate that CD226 is an antiapoptotic molecule and may play an important role in murine thymocyte development.