We have recently described a bone marrow culture system which is able to maintain, for at least 2 weeks, cells which have the capacity to repopulate the thymus of irradiated recipient mice (pre-T cells). Because this culture system depends upon the addition of an exogenous growth factor (IL-3) which may potentially influence the differentiation of the cultured pre-T cells, it is important to determine whether or not the progeny of cultured marrow cells are able to develop within the thymus in a kinetically normal fashion. Here we report the results of an analysis of the progeny of those cultured progenitor cells at 2, 3, and 4 weeks following intrathymic transfer. The passage of cultured donor-derived cells through critical early (expression of the IL-2 receptor) and late (expression of high levels of CD3) intrathymic events was assessed in these studies and compared with the pattern observed in the progeny of fresh bone marrow cells. The results of these studies showed that the progeny of cultured pre-T cells were able to develop expression of the IL-2 receptor and CD3 surface antigen during their residency within the thymus. In addition, both the timing and levels of expression of these surface markers were virtually identical on the progeny of fresh and cultured pre-T cells. These data suggest that cultured pre-T cells are not dramatically altered by their passage in vitro and are able to give rise to normally developing thymocytes upon in vivo transfer.