Induced regulatory T cells (iTreg) are a heterogeneous T-cell subset that is induced during an allo-response and down-regulates the immune response. iTreg are commonly characterized as CD4(+)CD25(high), CD4(+)CD25(high)FoxP3(+), CD4(+)CD25(high)CD127(-), or CD4(+)CD25(high)FoxP3(+)CD127(-) peripheral blood lymphocytes (PBL). In the present study, we investigated the overlap of these 4 phenotypically determined iTreg subsets in normal human individuals. PBL of 8 healthy individuals were incubated for 0 hours (Group 1) or for 16 hours in medium without (Group 2) or with phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Group 3). Thereafter, proportions of PBL with Treg phenotypes were determined using 4-color flow-cytometry. All 4 Treg subsets increased strongly during polyclonal stimulation (P < .001). After stimulation, combining 2 iTreg markers, 24% of stimulated CD4(+) PBL were CD25(high)FoxP3(+), 18% CD25(high)CD127(-), and 61% FoxP3(+)CD127(-). Combining 3 iTreg markers, only 18% of the polyclonally stimulated CD4(+) PBL were CD25(high)FoxP3(+)CD127(-). Importantly, the proportion of FoxP3(+)CD127(-) PBL increased with the quantity of CD25 on stimulated CD4(+) PBL and was highest in CD25(high) PBL (P = .002), emphasizing the relevance of CD25(high) as iTreg marker. Different iTreg phenotypes should not be used interchangeably because they define different iTreg subsets that overlap only in part. CD25(high) is the most relevant iTreg marker. These conclusions should be considered when studies and experiments involving iTreg phenotypes are compared.