Culture systems utilizing Notch1 signaling can be used for the in vitro development of T lineage cells at various differentiation stages. The most widely used of those systems is the OP9-DL1 system, which uses a mouse bone marrow stromal cell line transduced to express the Notch1 ligand Delta-like 1 (DL1) to coculture hematopoietic stem cells in the presence of IL-7 and FLT3-ligand. This system can be modified for the generation of large numbers of lymphoid progenitors committed to the T lineage for adoptive immunotherapy. We recently demonstrated that co-transplanted allogeneic OP9-DL1 derived early T progenitors can mature in an immunosuppressed host, mediating immunity including anti-tumor activity without causing GVHD [see selected publications], and can even be transferred in the absence of allogeneic stem cells to any immunosuppressed individual irrespective of MHC disparities for adoptive ‘off-the-shelf’ immunotherapy (see selected publications). Such cells also protect the thymus from atrophy which otherwise hinders future lymphopoiesis, and can be genetically modified in vitro for targeted immunotherapy. Notch-based culture systems show promise for clinically applicable therapeutic use: they can be fully humanized and human cord blood and human adult BM derived CD34+ progenitor cells have been cultured in these systems to generate human T cells.