The laboratory plays a central role in the evaluation of immune function and is critical in the diagnosis and treatment of immune deficiencies. The range of options available to evaluate lymphocyte function has expanded dramatically as our understanding of the immune system has expanded. As the choices of laboratory tests increase, so also does the need to choose testing in such a way as to appropriately direct the evaluation. Typically, this approach involves starting with screening tests and, on the basis of the results of these tests, deciding whether more sophisticated and expensive testing is warranted. The remarkable developments over the past decade leading to the identification of numerous gene defects underlying a variety of immune deficiencies has moved mutation analysis into the realm of the clinical laboratory. This information could be indispensable for immune deficiency diagnosis, prenatal screening, carrier detection, and family counseling. In this review a sequential approach to evaluating lymphocyte function is presented, starting with readily available screening tests and followed by more complex in vitro testing, including the application of newer assays. The various approaches are presented from the perspective of appropriate use and information garnered, whereas actual details of test procedures are not discussed but are referenced. The evolution of immune function testing suggests that it will continue to develop, and future assays are likely to provide even more insight into specific aspects of the immune response and be linked to immune deficiencies not yet defined.