<p>Human invariant natural killer T (iNKT) cells are a conserved population of innate-like T cells that are activated by glycolipid antigens. In addition to their well-known role in anti-tumor function, iNKT cells are also involved in regulating and maintaining hematopoiesis in the bone marrow. Here, we present the reprogramming of human CD4⁺Vα24⁺Vβ11⁺ iNKT cells into induced pluripotent stem cells (iNKT-iPSCs) and describe a novel chemically defined, feeder-free 3D spheroid method for generating CD34⁺ cells from iNKT-iPSCs, followed by their re-differentiation into functional Vα24⁺Vβ11⁺ iNKT cells (i-iNKT) with pro-hematopoietic activity. The i-iNKT cells showed specific binding to CD1d tetramers loaded with the lipid antigen α-galactosylceramide and had a similar transcription factor profile to that of somatic CD4⁺ iNKT cells. Additionally, in response to CD3 stimulation, the i-iNKT cells produced cytokines with hematopoietic potential and promoted expansion/differentiation of myeloid progenitors. These findings suggest the feasibility of using iPSCs as off-the-shelf i-iNKT cell sources to enhance the hematopoietic activity of bone marrow after hematopoietic stem cell (HSC) transplantation. </p>