CD4+CD25+ regulatory T cells (Tregs) are essential negative regulators of immune responses. Here, we examined the signaling properties of human Tregs, using CD4+CD25+ Treg and CD4+CD25+ control (Tcont) cell lines generated from cord blood. Treg cell lines were markedly hyporesponsive to stimulation with dendritic cells and with anti-CD3/CD28-coated beads. Hyporesponsiveness was reversed by exogenous interleukin-2 (IL-2). T-cell receptor (TCR)-CD3/CD28- mediated activation of Rap1 and Akt was retained in Tregs, but activation of Ras, mitogenactivated protein kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase 1/2 (Erk1/2) was impaired. Tregs were blocked from cell cycle progression due to decrease of cyclin E and cyclin A and increase of p27kip1 (p27kip cyclin dependent kinase inhibitor). IL-2 induced sustained increase of cyclin E and cyclin A and prevented up-regulation of p27kip1. Tregs had high susceptibility to apoptosis that was reversed by IL-2, which correlated with activation of Erk1/2, up-regulation of Bcl-XL (B-cell CLL/lymphoma 2-like nuclear gene encoding mitochondrial protein, transcript variant 2), and phosphorylation of Bad (Bcl2 antagonist of cell death) at Ser112. Thus, Tregs share biochemical characteristics of anergy, including abortive activation of Ras-MEK-Erk, increased activation of Rap1, and increased expression of p27kip1. In addition, our results indicate that TCR-CD3/CD28-mediated and IL-2 receptor-mediated signals converge at the level of MEK-Erk kinases to regulate Treg survival and expansion and suggest that manipulation of the MEK-Erk axis may represent a novel strategy for Treg expansion for immunotherapy.