Flow cytometric analysis of platelet structure and function provides useful information for both experimental and clinical applications. Flow cytometric assays have been developed to measure glycoprotein expression and activation markers on circulating platelets, agonist-specific reactivity, granule release markers, surface binding of secreted or plasma proteins, binding of platelets to other blood cells, procoagulant platelet-derived microparticles, changes in intracellular calcium levels, phosphorylation of signaling molecules, cytoskeletal rearrangement, and more. Important advantages of platelet flow cytometry over other platelet analysis methods include applicability to thrombocytopenic samples, low sample volume requirement, assessment of platelets in their native environment (whole blood), and the ability to measure multiple markers on each cell in a single test enabling characterization of platelet populations. Relevant clinical applications include assessment of thrombotic risk in cardiovascular diseases and cancer, monitoring of pharmacological antiplatelet therapy, and diagnosis of inherited and acquired platelet function disorders. The present chapter focuses on (1) methodological aspects of human platelet flow cytometry, including sample processing and markers/endpoints, (2) clinical applications of platelet flow cytometry assays, and (3) platelet flow cytometric assays in translational and basic research.