T cells are produced by the differentiation of hematopoietic stem and progenitor cells (HSPCs) into Lin− CD34+ CD7+ progenitors that migrate from the bone marrow (BM) to the thymus where further T cell development takes place. The differentiation of HSPCs into T cells can be supported in vitro by using OP9 stromal cells that express a Notch ligand. We have developed a more simple stroma-free culture method that promotes the expansion and differentiation of T cell progenitors from purified CD34+ HSPCs and purified multipotent lymphoid progenitors (MLPs) from cord blood (CB) or BM. CD34+ CB cells isolated using EasySep magnetic separation or CD34+ CD38− CD90− CD45RA+ MLPs further purified by cell sorting were seeded into culture plates coated with an attachment substrate containing Notch ligand and cultured in serum-free StemSpan SFEM II medium in the presence of a cytokine supplement containing SCF, TPO, Flt3L and IL-7. After 3 weeks, CD7+ CD5+ proT cells were detected at a frequency of 84% (range: 63–97%, n=26) with a proT cell yield of 2,130 (142–5,970, n=26) per initial CD34+ cell and 28,110 (4,720–63,930, n=4) per MLP initially seeded. In these cultures 28% (12–43%, n=12) of CD7+ cells also expressed CD1a, indicating further differentiation into preT cells. The pro and preT cells could mature into CD4+ CD8+ double positive T cells (38%; 24–64%, n=6) and TCRαβ+ CD3+ T cells (12%; 5–23%) over two weeks of continued culture in the same medium and in the presence of OP9-DL1 stromal cells. These results demonstrate that HSPCs can expand and differentiate into pro and preT cells under stroma-free and serum-free culture conditions. This novel culture system may benefit research and development of immunotherapies where large numbers of T cells are required.