The thymus is essential for proper development and maintenance of a broad T cell repertoire capable of recognizing a wide-range of foreign antigens. Recent advances in multicolor flow cytometry, non-invasive imaging techniques, and molecular assessments of thymic function have enabled a more comprehensive characterization of human thymic output in clinical settings than in the past. These techniques have been particularly valuable in monitoring human T cells after therapeutic thymic grafting for complete DiGeorge syndrome and during HIV-1 infection and AIDS. By defining the degree and mechanisms of T cell reconstitution in these settings, clinical investigators and primary caregivers have been able to better diagnose, treat and care for individuals with congenital or acquired immune deficiencies associated with loss of thymic function.