Flow cytometry and cell sorting are critical tools in stem cell research. Recent advances in flow cytometric hardware, reagents, and software have synergized to permit the stem cell biologist to more fully identify and isolate rare cells based on their immunofluorescent and light scatter characteristics. Some of these improvements include physically smaller air‐cooled lasers, new designs in optics, new fluorescent conjugate‐excitation pairs, and improved software to visualize data, all which combine to open up new horizons in the study of stem cells, by enhancing the resolution and specificity of inquiry. In this review, these recent improvements in technology will be outlined and important cell surface and functional antigenic markers useful for the study of stem cells described. © 2009 Clinical Cytometry Society