Foxp3 is regarded as the major transcription factor for T regulatory (Treg) cells and expression of Foxp3 is used to identify and quantitate Treg cells in mouse models. However, several studies have demonstrated that human CD4+T conventional (Tconv) cells activatedin vitroby T cell receptor (TCR) stimulation can express Foxp3. This observation has raised doubt as to the suitability of Foxp3 as a Tregmarker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3+Tregcells and can potentially serve as a marker of human Treg. Here, we confirm that Foxp3 expression is readily upregulated by Tconvupon TCR stimulationin vitro, while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hTconvin a humanized mouse model of graft versus host disease (GVHD) and in patients with a wide variety of acute and chronic inflammatory diseases including sickle cell disease, acute and chronic GVHD, systemic lupus erythematosus, as well as critical COVID-19. In all patients studied, an excellent correlation was observed between the percentage of CD4+T cells expressing Foxp3 and the percentage expressing Helios. Taken together, these studies demonstrate that Foxp3 is not induced upon Tconvcell activationin vivoand that Foxp3 expression alone can be used to quantitate Tregcells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of Tregin humans.