Foxp3(+)CD4(+) regulatory T cells (Tregs) are crucial in maintaining self-tolerance and limiting immune responses to pathogens. Shifting the sensitive balance between Tregs and effector T cells requires extensive knowledge of the homeostatic properties of the different T cell populations. For the investigation of Treg homeostatic expansion, we introduce in this study novel BAC transgenic mice, designated Foxp3.LuciDTR, coexpressing enhanced GFP, luciferase for bioluminescence imaging of Tregs, and the diphtheria toxin receptor (DTR) for specific ablation of Tregs. Of several founder lines, Foxp3.LuciDTR-4 mice displayed approximately 95% Treg depletion following injection of DT, resulting in activation of conventional CD4(+) T cells, probably due to lack of control by Tregs. In contrast, Foxp3.LuciDTR-3 mice displayed only approximately 70% Treg depletion without concomitant activation of CD4(+) T cells and represented, therefore, a suitable model to study Treg homeostasis in an environment where other T cell populations were not altered. After depletion, the Treg compartment recovered to its original size in approximately 2 wk. This recovery was mediated in a thymus-independent fashion by homeostatic proliferation of the surviving, nondepleted Tregs. The proliferating Tregs acquired an activated phenotype and maintained their suppressive capacity. Studies involving DT-mediated depletion of dendritic cells in CD11c.DOG mice showed that dendritic cells were required for optimal Treg homeostasis. In addition, IL-2 was identified as an essential factor for homeostatic recovery of the Treg compartment. These results show that Treg homeostasis is specifically regulated by the size of the Treg compartment and is independent of proliferation of conventional T cells.