CD4+CD25high CD127low/neg regulatory T cells (Tregs) play a critical role in the maintenance of peripheral tolerance and in controlling the development of autoimmune diseases. A combination of surface and intracellular markers, namely, CD25, CD39/CD73, CD62L, CD45RO, CD127, glucocorticoid-induced tumor necrosis factor receptor (GITR), CTLA-4, and the forkhead/winged helix transcription factor (FOXP3), has been used to characterize Tregs. Tregs suppress T effector responses mainly in a direct cell–cell contact manner. However, other mechanisms independent from this manner cannot be excluded entirely. It has been shown that Tregs can undergo limited expansion in vitro after the stimulation of TCR in the presence of exogenous cytokines, e.g., IL-2. Expanded Tregs retain their suppression function. Human Tregs have demonstrated their great potential to be used as a therapeutic intervention in preventing graft rejection and treating autoimmune diseases. In this chapter, we have given a review on how to characterize, isolate, expand Tregs and assess their suppressive functions.