This document provides an overview of flow cytometry techniques and applications. It discusses experimental controls used in flow cytometry including unstained, isotype, internal negative, positive, compensation and viability controls. It describes how flow cytometry can be used to analyze surface and intracellular markers, immune responses, cell proliferation, migration, suppression potential and more. Specific examples are given on analyzing peripheral blood mononuclear cells, lung tissue, lymph nodes and studying CD4+CD25+ T cell suppression and migration. Guidance is provided on reporting flow cytometry data, controlling for autofluorescence, validating results against literature, and properly analyzing the large amount of data generated.