Naive T cells undergo homeostatic proliferation in lymphopenic mice, a process that involves TCR recognition of specific self peptide/MHC complexes. Since costimulation signals regulate the T cell response to foreign Ags, we asked whether they also regulate homeostatic expansion. We report in this study that homeostatic expansion of CD4 and CD8 T cells occurs independently of costimulation signals mediated through CD28/B7, CD40L/CD40, or 4-1BB/4-1BBL interactions. Using DO11. 10 TCR transgenic T cells, we confirmed that CD28 expression was dispensable for homeostatic expansion, and showed that the presence of endogenous CD4+ CD25+ regulatory cells did not detectably influence homeostatic expansion. The implications of these findings with respect to regulation of T cell homeostasis and autoimmunity are discussed.