This is the author accepted manuscript. The final version will be available from Elsevier at http://dx.doi.org/10.1016/j.exphem.2015.05.006. ; Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation often performed using multi-parameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolving the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AADdim fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data show that, when used in combination with single cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single cell systems, both for improving isolation and for acquiring additional cell surface marker information. ; This work was supported by grants from Leukaemia and Lymphoma Research, the Medical Research Council, the National Institute for Health Research Cambridge Biomedical Research Centre, and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust?MRC Cambridge Stem Cell Institute. DGK is the recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship and a European Hematology Association non-clinical advanced research fellowship. The authors declare that they have no conflict of interest.