ABSTRACTAchievement of immunocompetent and therapeutic T lymphopoiesis from pluripotent stem cells is a central aim in T cell regenerative medicine. To date, preferentially regenerating T lymphopoiesis in vivo from pluripotent stem cells (PSC) remains a practical challenge. Here we documented that synergistic and transient expression of Runx1 and Hoxa9 restricted in the time window of endothelial to hematopoietic transition and hematopoietic maturation stages induced in vitro from PSC (iR9-PSC) preferentially generated engraftable hematopoietic progenitors capable of homing to thymus and developing into mature T (iT) cells in primary and secondary immunodeficient recipients. Single-cell transcriptome and functional analyses illustrated the cellular trajectory of T lineage induction from PSC, unveiling the T-lineage specification determined at as early as hemogenic endothelial cell stage and identifying the bona fide pre-thymic progenitors. The iT cells distributed normally in central and peripheral lymphoid organs and exhibited abundant TCRαβ repertoire. The regenerative T lymphopoiesis rescued the immune-surveillance ability in immunodeficient mice. Furthermore, gene-edited iR9-PSC produced tumor-specific-T cells in vivo that effectively eradicated tumor cells. This study provides insight into universal generation of functional and therapeutic T lymphopoiesis from the unlimited and editable PSC source.