Recent technological advancements have enabled the flow cytometric measurement of tens of parameters on millions of cells. Conventional manual data analysis and bioinformatics tools cannot provide a complete analysis of these datasets due to this complexity. In this chapter we will provide an overview of a general data analysis pipeline both for automatic identification of cell populations of known importance (e.g., diagnosis by identification of predefined cell population) and for exploratory analysis of cohorts of flow cytometry assays (e.g., discovery of new correlates of a malignancy). We provide three real-world examples of how unsupervised discovery has been used in basic and clinical research. We also discuss challenges for evaluation of the algorithms developed for (1) identification of cell populations using clustering, (2) identification of specific cell populations, and (3) supervised analysis for discriminating between patient subgroups.