Natural regulatory T cells (nTreg) can suppress different immune‐cell responses and maintain the balance between tolerance and immunity in the individual. These cells are defined by CD4⁺, CD25^hi, and FOXP3⁺ expression, although a variety of other nTreg‐associated markers have been reported to be expressed at different levels (e.g., HLA‐DR, CTLA‐4, GARP, Helios, CD39, etc.), presumably reflecting different functional stages of the heterogeneous nTreg population. Several markers show low/negative expression (i.e., CD127, CD49d, and CD26), but none of these markers are specific to nTreg. CD25^hi expression has been a useful surface marker to identify/isolate nTreg; however, CD25 is also expressed on “adaptive” or “induced” Treg, as well as in activated conventional T cells. In addition, the fact that FOXP3 is also found in CD25 low/negative CD4⁺ T cells, and in a subset of CD8⁺ T cells, further complicates the definition of a specific nTreg marker. Although CD4⁺, CD25^hi, and CD127^low/negative markers characterize the majority of nTreg, it is still imperative to find additional surface‐marker combinations that improve the identification/isolation of nTreg and their subsets. Herein, we present evidence that CD4⁺CD25^hi CD6^lo/− nTreg have high expression of FOXP3and exhibit in vitro suppressive activity on CD8⁺ T‐cell proliferation. Dot‐plot analyses of CD4⁺ cells, with CD6, CD127, CD49d, or CD26 reveal that a higher enrichment yield of CD25^hiFOXP3⁺ cells can be achieved in the combined CD6^lo/−CD127^lo/− population. We conclude that FOXP3⁺ nTreg cells are characterized by CD6^lo/− expression, providing a new tool for the identification of nTreg cells without recourse to intracellular staining, and for the purification of these cells by negative selection. © 2014 International Society for Advancement of Cytometry