To develop a comprehensive catalogue of phenotypic and functional parameters of human CD4⁺ T cell differentiation stages, we have performed microarray gene expression profiling on subpopulations of human thymocytes and circulating naive CD4⁺ T cells, including CD3⁻CD4⁺CD8⁻ intrathymic T progenitor cells, CD3^intCD4⁺CD8⁺ ‘double positive’ thymocytes, CD3^highCD4⁺CD8⁻ ‘single positive’ thymocytes, CD3⁺CD4⁺CD8⁻ CD45RA⁺CD62L⁺ naive T cells from cord blood and CD3⁺CD4⁺CD8⁻ CD45RA⁺CD62L⁺ naive T cells from adult blood. These subpopulations were sort-purified to >98% purity and their expressed RNAs were analyzed on Affymetrix Human Genome U133 arrays. Comparison of gene expression signals between these subpopulations and with early passage fetal thymic stromal cultures identify: (i) transcripts that are preferentially expressed in human CD4⁺ T cell subpopulations and not in thymic stromal cells; (ii) major shifts in gene expression as progenitor T cells mature into progeny; (iii) preferential expression of transcripts at the progenitor cell stage with plausible relevance to the regulation of expansion and differentiation of these cells; and (iv) preferential expression of potential markers of recent thymic emigrants in naive-phenotype CD4⁺ T cells from cord blood. Further evaluation of these findings may lead to a better definition of human thymopoiesis as well as to improved approaches to monitor and to augment the function of this important organ of T cell production.