We have recently shown that endogenous Annexin-A1 (AnxA1) plays a homeostatic regulatory role in mature T cells by modulating the strength of TCR signaling. In this study we investigated the role of endogenous AnxA1 in thymocyte maturation. Analysis of AnxA1(-/-) thymocyte populations at the immature CD4(-)CD8(-) double negative (DN) stage showed a proportional decrease in the DN1 and an increase in the DN3 subsets compared to control littermates. There were no significant differences in thymocyte numbers or proportions of CD4(+) and CD8(+) single positive (SP) populations between Anx1(-/-) and AnxA1(+/+) mice. However, when we crossed AnxA1(-/-) mice onto HY-TCR transgenic mice, we observed an increase in CD4(+)CD8(+) double positive (DP) and CD4 SP cells in male AnxA1(-/-)/HY-TCR compared to AnxA1(+/+)/HY-TCR. Conversely, female AnxA1(-/-)/HY-TCR mice showed an increase in DP and a decrease in CD8 (SP) cells compared to female AnxA1(+/+)/HY-TCR. Biochemical analysis of the signaling pathways responsible for these effects showed a decrease in anti-CD3-induced Erk phosphorylation and NFkappaB activation in AnxA1(-/-) thymocytes compared to control littermates. Together these findings demonstrate a role for endogenous AnxA1 in regulating both positive and negative selection of the TCR repertoire. These results suggest that targeting AnxA1 expression or function in T cells could represent a useful approach for the development of novel therapies for the treatment of autoimmune diseases.