Authenticating markers for the functional suppressive CD4⁺FoxP3⁺ regulatory T cells (Tregs) are important for the quantitative identification and enrichment of viable Tregs for possible therapeutic use. CD25 as a surrogate marker of Tregs has some limitations, which prompted investigators to identify more specific marker(s) of Tregs. The search for a firm molecular definition of Tregs resulted in the identification of FoxP3 as a better marker of this subset of CD4 cells. Nevertheless, FoxP3⁺ Tregs are phenotypically and functionally heterogeneous. Even in normal mice, only a minority of FoxP3⁺ T cells are potent suppressor cells. Therefore, additional marker(s) are required for delineation of truly functional Tregs. In this review, the studies identifying markers of functional Tregs, both in mouse and in human, and their functional implications are discussed. Our finding that TNFR2, which mediates the effect of TNF on the activation of Tregs, is a superb marker of the most suppressive subset of mouse Tregs and its application in the identification of functional human Tregs will also be reviewed.