Transforming growth factor-β (TGF-β) maintains self-tolerance through a constitutive inhibitory effect on T-cell reactivity. In most physiological situations, the tolerogenic effects of TGF-β depend on the canonical signaling molecule Smad3. To characterize how TGF-β/Smad3 signaling contributes to maintenance of T-cell tolerance, we characterized the transcriptional landscape downstream of TGF-β/Smad3 signaling in resting or activated CD4 T cells. We report that in the presence of TGF-β, Smad3 modulates the expression of >400 transcripts. Notably, we identified 40 transcripts whose expression showed Smad3 dependence in both resting and activated cells. This 'signature' confirmed the non-redundant role of Smad3 in TGF-β biology and identified both known and putative immunoregulatory genes. Moreover, we provide genomic and functional evidence that the TGF-β/Smad3 pathway regulates T-cell activation and metabolism. In particular, we show that TGF-β/Smad3 signaling dampens the effect of CD28 stimulation on T-cell growth and proliferation. The impact of TGF-β/Smad3 signals on T-cell activation was similar to that of the mTOR inhibitor Rapamycin. Considering the importance of co-stimulation on the outcome of T-cell activation, we propose that TGF-β-Smad3 signaling may maintain T-cell tolerance by suppressing co-stimulation-dependent mobilization of anabolic pathways.Genes and Immunity advance online publication, 17 January 2013; doi:10.1038/gene.2012.63.