Regulatory T (Treg) cells play a key role in dominant suppression of immune response and maintenance of immune homeostasis. Foxp3, a member of the forkhead transcription factor family, is indispensable for Treg cell development and function. Mice and human with Foxp3 mutations are severely impaired in Treg cell generation and develop lethal autoimmune diseases. We combined chromatin immuno-precipitation and mouse whole genome tiling array profiling (ChIP-on-Chip) to identify the direct downstream targets of Foxp3 in regulatory T cells. Our result showed that Foxp3 not only directly determines expression of a number of Treg signature molecules, but also regulates a group of transcription factors, which potentially control the expression of other Treg-specific genes.