Differentiation of mouse embryonic stem cells (ESCs) or hematopoietic stem cells (HSCs) from fetal liver or bone marrow into T-lymphocytes can be achieved in vitro with the support of OP9-DL1 cells, a bone-marrow-derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (Dll1). This approach provides a simple, versatile, and efficient culture system that allows for the commitment, differentiation, and proliferation of T-lineage cells from different sources of stem cells. This article contains a series of protocols, the first of which describes the establishment, maintenance, and storage of OP9 and OP9-DL1 cells. Subsequent protocols detail how to co-culture the OP9 and OP9-DL1 cells with either ESCs or HSCs from fetal liver or bone marrow, leading to in vitro differentiation of the stem cells into lymphocytes.