The mechanisms by which prolonged estrogen exposures, such as estrogen therapy and pregnancy, reduce thymus weight, cellularity, and CD4 and CD8 phenotype expression, have not been well defined. In this study, the roles played by the membrane estrogen receptor, G protein-coupled receptor 30 (GPR30), and the intracellular estrogen receptors, estrogen receptor · (ER) and (ER), in 17-estradiol (E2)-induced thymic atrophy were distinguished by construction and the side-by-side comparison of GPR30-deficient (GPR30KO) mice with ER (AERKO) and ER (BERKO) gene-deficient mice. Our study shows that while ER mediated exclusively the early developmental blockage of thymocytes, GPR30 was indispensable for thymocyte apoptosis that preferentially occurs in TCR^-/low DP thymocytes. Additionally, G1, a specific GPR30 agonist, induces thymic atrophy and thymocyte apoptosis, but not developmental blockage. Finally, E2-treatment attenuates the activation of NFB in CD25^-CD4^-CD8^- double negative (DN) thymocytes through an ER · -dependent yet ER- and GPR30-independent pathway. Differential inhibition of NFB by ER and GPR30 might underlie their disparate physiological effects on thymocytes. Our study distinguishes for the first time the respective contributions of nuclear and membrane E2 receptors in negative regulation of thymic development.