AbstractThe generation of T-cells from stem cells in vitro could provide an alternative source of cells for immunotherapies. T-cell development from hematopoietic stem and progenitor cells (HSPCs) is tightly regulated through Notch pathway activation by Delta-like (DL) ligands 1 and 4. Other molecules, such as stem cell factor (SCF) and interleukin (IL)-7, play a supportive role in regulating the survival, differentiation, and proliferation of developing T-cells. Numerous other signaling molecules influence T-lineage development in vivo, but little work has been done to understand and optimize their use for T-cell production. Using a defined engineered thymic niche system, we undertook a multi-stage statistical learning-based optimization campaign and identified IL-3 and tumor necrosis factor α (TNFα) as a stage- and dose-specific enhancers of cell proliferation and T-lineage differentiation. We used this information to construct an efficient three-stage process for generating conventional TCRαβ⁺CD8⁺T-cells expressing a diverse TCR repertoire from blood stem cells. Our work provides new insight into T-cell development and a robust system for generating T-cells to enable clinical therapies for treating cancer and immune disorders.