Naive peripheral CD4(+)CD25(-) T cells can be converted into Foxp3-expressing regulatory T cells under appropriate stimulation conditions. Considering that continuous exposure to antigens is one of the prerequisites for the differentiation and maintenance of Treg cells, we investigated whether preventing activation-induced cell death while providing continuous TCR stimulation could promote the expression of Foxp3 in murine naive CD4(+) T cells. Among the several anti-apoptotic agents tested, aurintricarboxylic acid (ATA) was found to induce the in vitro conversion of naive CD4(+) T cells into Foxp3(+) Treg cells with suppressive activity. Neutralizing studies with an antibody against transforming growth factor (TGF)-β revealed that ATA requires the presence of TGF-β to induce Foxp3 expression in naive CD4(+)CD25(-) T cells. Although ATA itself did not activate the Smad signaling pathway, it down-regulated the extracellular signal-regulated kinase and mammalian target of rapamycin signaling cascade in activated T cells. Lastly, combined exposure to ATA and TGF-β had a synergistic effect on the rate of induction and maintenance of Foxp3 expression. These results indicate that ATA could be exploited to efficiently prepare inducible regulatory T cells in vitro and may aid in more precisely identifying the specific signaling pathways that drive Foxp3 expression in T cells.