Although recent in vitro studies have begun to decipher the molecular events that characterize the anergic state, their in vivo biologic relevance and potential clinical importance remain unclear. Here, using anergic human T-cell clones and tolerant alloreactive mouse T cells that do not induce graft-versus-host disease, we show that p27 kip1 cyclin-dependent kinase inhibitor is an essential regulator responsible for the blockade of clonal expansion of anergic T cells in vitro and in vivo. Moreover, in anergic cells, p27 kip1 associates with the c-Jun co-activator JAB1, resulting in defective transactivation of AP-1 and interleukin 2 transcription. Therefore, pharmacological agents that upregulate the expression of or prevent the degradation of p27 kip1 during antigen recognition should be part of new therapeutic strategies to induce antigen-specific T-cell unresponsiveness.