Immunophenotyping of blood lymphocyte subpopulations is an important tool in the diagnosis of immunological and hematological diseases. Pediatric age-matched reference values have been determined for the major lymphocyte populations, but reliable reference values for the more recently described T-lymphocyte subpopulations, like different types of memory T-lymphocytes, recent thymic emigrants, regulatory T-cells and CXCR5 + helper-Tlymphocytes, are not sufficiently available yet. We determined reference values for the absolute and relative sizes of T-lymphocyte subpopulations in healthy children using the lysed whole blood method, which is most often used in diagnostic procedures. When the absolute numbers of some or all T-lymphocyte subpopulations fall outside these reference ranges, this may indicate disease. The reference values show the course of T-lymphocyte development in healthy children. Absolute T-lymphocyte numbers increase 1.4-fold during the first months of life and after 9-15 months they decrease threefold to adult values; this is mainly caused by the expansion of recent thymic emigrants and naive cells. Helper and cytotoxic T-lymphocytes show the same pattern. Regulatory T-cells increase in the first five months of life and then gradually decrease to adult values, although the absolute numbers remain small. The relative number of CXCR5 + cells within the CD4 + CD45RO + T lymphocytes increases during the first six months of life and then remains more or less stable around 20%.and negative control of immune responses. Annu Rev Immunol. 2004;22:531-62. 11 Kim CH, Rott LS, Clark-Lewis I, Campbell DJ, Wu L, Butcher EC. Subspecialization of CXCR5+ T cells: B helper activity is focused in a germinal center-localized subset of CXCR5+ T cells. 13 van Gent R, van Tilburg CM, Nibbelke EE et al. Refined characterization and reference values of the pediatric T-and B-cell compartments. Clin Immunol. 2009 Oct;133:95-107. 14 Reimann KA, Chernoff M, Wilkening CL, Nickerson CE, Landay AL. Preservation of lymphocyte immunophenotype and proliferative responses in cryopreserved peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected donors: implications for multicenter clinical trials. The ACTG Immunology Advanced Technology Laboratories. Clin Diagn Lab Immunol. 2000 May;7:352-9. 15 Hokland P, Heron I. Analysis of the lymphocyte distribution during Isopaque-Ficoll isolation of mononuclear cells from human peripheral blood. J Immunol Methods. 1980;32:31-9. 16 Saule P, Trauet J, Dutriez V, Lekeux V, Dessaint JP, Labalette M. Accumulation of memory T cells from childhood to old age: central and effector memory cells in CD4(+) versus effector memory and terminally differentiated memory cells in CD8(+) compartment. Mech Ageing Dev. 2006 Mar;127:274-81. 17 Kraan J, Gratama JW, Keeney M, D'Hautcourt JL. Setting up and calibration of a flow cytometer for multicolor immunophenotyping.