Stem cell mechanics, determined predominantly by the cell’s cytoskeleton, plays an important role in different biological processes such as stem cell differentiation or migration. Several methods to measure mechanical properties of cells are currently available, but most of them are limited in the ability to screen large heterogeneous populations in a robust and efficient manner—a feature required for successful translational applications. With real-time fluorescence and deformability cytometry (RT-FDC), mechanical properties of cells in suspension can be screened continuously at rates of up to 1,000 cells/s—similar to conventional flow cytometers—which makes it a suitable method not only for basic research but also for a clinical setting. In parallel to mechanical characterization, RT-FDC allows to measure specific molecular markers using standard fluorescence labeling. In this chapter, we provide a detailed protocol for the characterization of hematopoietic stem and progenitor cells (HSPCs) in heterogeneous mobilized peripheral blood using RT-FDC and present a specific morpho-rheological fingerprint of HSPCs that allows to distinguish them from all other blood cell types.