Flow cytometry is an incredibly powerful diagnostic tool in the evaluation of primary and secondary immune deficiencies. Assay design and setup involves a methodological consideration of specimen collection, marker and fluorochrome selection, antibody titration, instrumentation, compensation, gating, reference range development, and cross validation. Commonly used analyses for lymphocytes are the lymphocyte subset, T-cell subset, B-cell and T-cell naive/memory, double-negative T-cell, and plasmablast panels. Flow cytometry has direct clinical applicability to the workup of severe forms of primary immune deficiency disorders and is used diagnostically and for therapeutic monitoring in the context of secondary immune deficiency disorders.