This unit illustrates the challenges associated with measurements of T cell subpopulations, and describes how those challenges can be overcome by using polychromatic (5+ color) approaches to flow cytometry. Such approaches are immensely powerful and directly applicable to studies of T cell biology in HIV disease; however, their development requires careful consideration of the antibodies and fluorochromes employed, as discussed here. Furthermore, factors generally known to influence T cell counts can also govern the success or failure of polychromatic experiments; therefore, these factors are reviewed and provide recommendations for minimizing their influence on polychromatic measurements of T cell subsets.