The differentiation of T lymphocyte precursors into functionally mature progeny proceeds in distinct stages. Since these are identified by characteristic constellations of phenotypic markers, the effects of experimental manipulations on T cell development can be readily monitored. In order to complete their developmental program, thymocytes must interact with stromal elements, which positively and negatively select the functional T cell receptor (TCR) repertoire. This process normally assures self tolerance and immunocompetence. Disturbances are of practical importance for clinical disorders including immunodeficiencies and autoimmune phenomena, raising a particular interest in human T cell development and repertoire formation. Here, we discuss results and possible applications of a culture system for human thymocytes. Further, we describe an in vitro approach addressing the requirements for a crucial step in T cell development; the transition from the immature CD4 CD8 double positive (DP) to the mature CD4 single positive (SP) stage.