Early studies of mice subjected to neonatal thymectomy and analyses of adoptive T-cell transfer into lymphopenic hosts led to the identification of a specialized subset of regulatory CD4+ T cells capable of suppressing various manifestations of autoimmunity. Recently, a combination of genetic, molecular, and traditional cellular approaches provided novel powerful means to investigate the biology of these cells. Here, we review earlier and current work from our laboratory, establishing a dedicated function for the transcription factor Foxp3 in the process of regulatory T-cell lineage commitment and a role for TCR- and cytokine-mediated signals in regulation of Foxp3 expression.