Mature hematopoietic cells, like all other terminally differentiated lineages, arise during ontogeny via a series of increasingly restricted intermediates. Hematopoietic progenitors have their origin in the mesoderm, which gives rise to hemangioblasts that can differentiate into endothelial or endocardial precursors or hematopoietic stem cells. These hematopoietic stem cells 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 environment in vitro has facilitated our study of this complex process. Until recently, the T-lymphocyte lineage was the exception and appeared to require the specialized three-dimensional microenvironment of the thymus to develop. However, here we describe a protocol for the generation of T lymphocytes from embryonic stem cells in vitro, within a two-dimensional microenvironment, provided by OP9 bone marrow stromal cells. This procedure will facilitate further study of early T lymphopoiesis by providing a simple model system in which the effects of genetic and environmental manipulations of embryonic stem cell-derived progenitors can be examined without requiring other more complex in vivo or in vitro experimental approaches.