AbstractWe and others have previously reported that thymic epithelial cells produce glucocorticoids (GCs). In vitro studies have also suggested that thymic-derived GCs play a role in the development of thymocytes. However, until now it has not yet been established whether thymic-derived GCs play a role in thymopoiesis in vivo. To investigate this, we conditionally overexpressed the GC receptor (GR) in thymocytes using transgenic mice with a tetracycline-inducible expression system. The influence of systemic GCs was excluded by adrenalectomizing the transgenic mice before the GR induction. Conditional expression of transgenic GR in the thymocytes of adrenalectomized transgenic mice led to a decrease in the thymocyte number. This was associated with increased thymocyte apoptosis. The effect of thymic-derived GCs on the thymocytes was confirmed after transgenic GR induction in a thymic organ culture system. Finally, the GR antagonist RU486 increased thymocyte number in adrenalectomized mice in vivo and prevented a reduction in thymocyte number in thymic organ culture after transgenic GR induction. These observations further confirmed a role for the thymic-derived GCs in regulating thymocyte homeostasis in vivo.