Regulatory T (Treg) cells are characterized by the expression of CD4, CD25 and the intracellular Foxp3. However, these markers do not indicate whether Treg cells are thymic derived Treg (tTreg) cells or peripherally induced Treg (pTreg) cells. Recently, Helios and Neuropilin-1 (Nrp1) has been reported as potential markers for tTreg cells. Herein, we used flow cytometry to examine the proportion of CD4⁺CD8⁻CD25⁺ Treg cells expressing Helios, Nrp1 and Foxp3 in thymus, pancreatic draining lymph nodes (PDLNs) and spleen of CD-1 mice and thymus of NOD and C57BL/6 mice. The frequency of Helios⁺ cells was higher than that of Nrp1⁺ cells in CD4⁺CD8⁻CD25⁺ and CD4⁺CD8⁻CD25⁺Foxp3⁺ Treg cells in thymus. Interestingly, the proportion of IL-10⁺, Ebi3⁺and CTLA-4⁺ cells was higher in Helios⁺ than Nrp1⁺ tTreg cells. The anti-apoptotic activity of Helios⁺ tTreg cells was higher in thymus compared to Nrp1⁺ tTreg cells. Nrp1 seems to be expressed at a later developmental stage compared to Helios and Foxp3. Furthermore, the expression of Nrp1 in CD4⁺CD25⁺ T cells of younger mice did not increase after stimulating them in vitro with anti-CD3 and –CD28. Thus, under these conditions, Helios could be considered a more reliable marker for distinguishing tTreg cells from pTreg cells than Nrp1.