This protocol describes a simple in vitro artificial thymic organoid (ATO) culture method for the differentiation of mature, naïve CD3+TCRαβ+ and TCRγδ+ T cells from hematopoietic stem and progenitor cells (HSPCs). This protocol accompanies Seet et al (Nature Methods, published online 3 April, 2017; 10.1038/nmeth.4237); it was added to the manuscript after formal peer review, as an aid to users. ATOs use standardized, off-the-shelf components and a novel serum-free media formulation to maximize the efficiency of T cell differentiation and minimize experimental variability. We have validated the ATO method for the generation of mature, naïve T cells from human cord blood, bone marrow, and mobilized or resting peripheral blood HSPCs, as well as purified hematopoietic stem cells and lymphoid progenitors. Furthermore, this method can be used for the differentiation of naïve antigen-specific T cells from TCR-transduced HSPCs. ATOs recapitulate thymic-like early T cell commitment, differentiation through all known progenitor and precursor stages of human CD3+TCRαβ+ thymopoiesis, and permit the positive selection and maturation of naïve CD3+TCRαβ+ single positive CD8+ and CD4+ T cells. ATOs are maintained with simple media changes, and do not require replenishment of stromal cells. The ATO system has direct applications for experimental studies of human T cell differentiation and stem-cell based approaches to T cell engineering.