ABSTRACT Foxp3 + regulatory T cells (Treg) are critical for the maintenance of self‐tolerance, and their absence or dysfunction can result in autoimmunity. To determine the critical cell type controlled by Treg and potentially the suppressor mechanism utilized by Treg in the steady state, we utilized mice expressing the diphtheria toxin receptor (DTR) exclusively on Treg cells. Complete depletion of Treg was achieved 24 h after DT treatment, but profound activation of CD4 + and CD8 + T cells as measured by induction of CD44 expression and proliferation required 3–4 days. Increased expression of CD80/CD86 was observed on dendritic cells and more prominently on macrophages after 3 days. Depletion of CD4 + T cells or macrophages resulted in ∼50% inhibition of T‐cell activation. The initial steps in T‐cell activation were completely independent of IFN‐γ or IL‐2, while upregulation of CD80/CD86 was partially dependent on IFN‐γ. Complete reversal of immune activation post‐Treg depletion was only achieved by blockade of CD80/CD86 interactions with CD28. We conclude that the major mechanism used by Treg in the steady state is the regulation of CD80/CD86 expression and dysregulation of this suppressor pathway results in lethal autoimmunity driven by co‐stimulatory signals in concert with TCR stimulation, or even by costimulatory signals alone.