Regulatory T (Treg) cells differentiate from thymocytes or peripheral T cells in response to host and environmental cues, culminating in induction of the transcription factor forkhead box P3 (Foxp3) and the Treg cell-specific epigenome. An intermediate amount of antigen stimulation is required to induce Foxp3 expression by engaging T cell receptor (TCR)-activated [e.g., nuclear factor (NF)-κB] and TCR-inhibited (e.g., Foxo) transcription factors. Furthermore, Treg cell differentiation is associated with attenuated Akt signaling, resulting in enhanced nuclear retention of Foxo1, which is indispensable for Treg cell function. These findings reveal that Treg cell lineage commitment is not only controlled by genetic and epigenetic imprinting, but also modulated by transcriptional programs responding to extracellular signals.