To delineate the cellular targets and mechanisms by which glucocorticoids (GCs) exert their actions, we generated mice in which a green fluorescent protein (GFP)-GC receptor (GR) fusion gene is knocked into the GR locus. In these mice, the GFP-GR protein, which is functionally indistinguishable from endogenous GR, allows the tracking and quantitation of GR expression in single living cells. In GFP-GR thymus, GR expression is uniform among embryonic thymocyte subpopulations but gradually matures over a 3-wk period after birth. In the adult, GR is specifically induced to high levels in CD25+CD4-CD8- thymocytes and returns to basal levels in CD4+CD8+ thymocytes of wild-type and positively selecting female HY TCR-transgenic mice, but not negatively selecting male HY TCR-transgenic mice. In GFP-GR/recombinase-activating gene 2-/- thymocytes, GR expression is down-regulated by pre-TCR complex stimulation. Additionally, relative GR expression is dissociated from GC-induced apoptosis in vivo. Results from these studies define differential GR expression throughout ontogeny, suggest pre-TCR activation as a specific mechanism of GR down-regulation, define immature CD8+ thymocytes as novel apoptosis-sensitive GC targets, and separate receptor abundance from susceptibility to apoptosis across thymocyte populations.