Notch family receptors control critical events in the production and replenishment of specialized cells in the immune system. However, it is unclear whether Notch signaling regulates abrupt binary lineage choices in homogeneous progenitors or has more gradual influence over multiple aspects of the process. A recently developed coculture system with Delta 1-transduced stromal cells is being extensively used to address such fundamental questions. Different from fetal progenitors, multiple types of adult marrow cells expanded indefinitely in murine Delta-like 1-transduced OP9 cell cocultures, progressed to a DN2/DN3 thymocyte stage, and slowly produced TCR(+) and NK cells. Long-term cultured cells of this kind retained some potential for T lymphopoiesis in vivo. Adult marrow progressed through double-positive and single-positive stages only when IL-7 concentrations were low and passages were infrequent. Lin(-)c-Kit(low)GFP(+)IL-7Ralpha(+/-) prolymphocytes were the most efficient of adult bone marrow cells in short-term cultures, but the assay does not necessarily reflect cells normally responsible for replenishing the adult thymus. Although marrow-derived progenitors with Ig D(H)-J(H) rearrangements acquired T lineage characteristics in this model, that was not the case for more B committed cells with V(H)-D(H)J(H) rearrangement products.