The induction of anergy in T cells, although widely accepted as critical for the maintenance of tolerance, is still poorly understood at the molecular level. Recent evidence demonstrates that in addition to blockade of costimulation using monoclonal antibodies (mAbs) directed against cell surface determinants, treatment of mixed lymphocyte reaction (MLR) cultures with interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) results in induction of tolerance, rendering alloreactive murine CD4+ T cells incapable of inducing graft-versus-host disease (GVHD) after in vivo transfer to histoincompatible recipients. The present study, using these cells prior to adoptive transfer, determined that IL-10 + TGF-β-tolerant CD4+ T cells exhibit an altered pattern of T-cell receptor (TCR) + CD28-mediated signaling and are incapable of progressing out of the G1 phase of the cell cycle during stimulation with HLA class II disparate antigen-presenting cells. TGFβ + IL-10-tolerant cells were incapable of phosphorylating TCR-ζ, or activating ZAP-70, Ras, and MAPK, similarly to T-cell tolerized by blockade of B7/CD28 and CD40/CD40L pathways. Moreover, these cells were incapable of clonal expansion due to defective synthesis of cyclin D3 and cyclin A, and defective activation of cyclin-dependent kinase (cdk)4, cdk6, and cdk2. These cells also exhibited defective down-regulation of p27kip1 cdk inhibitor and lack of cyclin D2-cdk4 activation, Rb hyperphosphorylation, and progression to the S phase of the cell cycle. These data link anergy-specific proximal biochemical alterations and the downstream nuclear pathways that control T-cell expansion and provide a biochemical profile of IL-10 + TGF-β-tolerant alloreactive T cells that do not induce GVHD when transferred into MHC class II disparate recipients in vivo.