We have developed a multi-parameter flow cytometry assay for the characterization of human regulatory T-cells. TBD Human FOXP3 Treg Characterization Kit includes three directly conjugated antibodies: CD3 PE/Cy5, CD4 FITC, and FOXP3 Alexa Fluor® 647, along with optimized fixation, permeabilization, wash, and assay buffers to provide researchers the ability to phenotypically distinguish human regulatory T-cells. Our combination of directly conjugated antibodies and optimized buffers allow for detection of CD3 and CD4 on the surface as well as FOXP3 within the nucleus simultaneously.