Summary Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for a variety of malignancies and other hematological disorders, and also holds promise for treatment of infectious diseases such as HIV. However, HSCT is limited by high risks of morbidity and mortality from complications such as graft failure (which leads to high susceptibility to lethal infections) and leukemic relapse. New strategies to improve outcomes of hematopoietic transplantation would thus have a significant clinical impact. This project focuses on developing invariant Natural Killer T (iNKT) cells derived from human induced pluripotent stem cells (iPSCs) as an adjunct HSCT cellular therapy designed to promote successful hematopoietic engraftment. In recently published preliminary studies, we showed that addition of allogeneic human CD4+ iNKT cells to human umbilical cord blood transplants led to dramatically improved hematopoietic engraftment in immunodeficient mice. The engraftment-promoting activity of iNKT cells was due to their interactions with cord blood monocytes that resulted in production of several potently pro-hematopoietic factors, including the cytokines GM-CSF and IL-3 (produced by iNKT cells), and PGE2 (produced by monocytes in response to signals from iNKT cells). Prior studies in murine models have established that cytokine programs and functional properties of iNKT cells are controlled by two key transcription factors, PLZF and E4BP4. How these transcription factors influence the cytokine profiles of human iNKT cells remains unclear, and specifically, their impact on iNKT cell production of GM-CSF and IL-3 and interactions with monocytes is not known. Aim 1 of this project proposes to generate human iPSC-derived iNKT cells lacking or over-expressing PLZF or E4BP4. Aim 2 will determine the impact of these transcription factors on the ability of iPSC-derived iNKT cells to promote human hematopoietic engraftment and on graft-versus-lymphoma (GVL) activity. Through these studies we will gain highly novel insight into the impact of PLZF and E4BP4 expression on iNKT functional properties, and we will establish the feasibility of generating iPSC-derived iNKT cells with modified transcription factor expression designed to stabilize specific functional programs. This will lay a foundation for designing iPSC-derived iNKT cells that are transcriptionally tuned to promote hematopoietic engraftment without adversely affecting GVL.