Mature hematopoietic cells, like all other terminally differentiated lineages, arise during ontogeny via a series of increasingly restricted intermediates. Hematopoietic progenitors derive from the mesoderm, which gives rise to hemangioblasts that can differentiate into endothelial or endocardial precursors, or hematopoietic stem cells (HSCs). These HSCs, in turn, may either self-renew or differentiate into lineage-restricted progenitors, and ultimately mature effector cells. The ability to generate most hematopoietic lineages in a two-dimensional in vitro environment has facilitated our study of this complex process. Until recently, T lymphocytes were the exception, and appeared to require the specific three-dimensional microenvironment of the thymus to develop. However, here we describe a protocol for the generation of immunocompetent T lymphocytes from embryonic stem cells (ESCs) in vitro, within the two-dimensional microenvironment provided by OP9 bone marrow stromal cells that have been transduced to express the Notch ligand Delta-like-1. This procedure will facilitate further study of T lymphocytes by providing a model system in which the effects of genetic and environmental manipulations of ESC-derived progenitors can be examined, and the mechanisms of tolerance potentially dissected, in vitro.