Despite their small numbers (~0.001-1% in blood), invariant natural killer T (iNKT) cells in humans have been suggested to play important roles regulating multiple diseases including infections, allergies, cancer and autoimmunity. Like all other immune cells, iNKT cells are derived from the blood stem cells living in the bone marrow of adult humans. Successful clinical interventions with iNKT cells have been greatly hindered by our limited knowledge on how these cells are produced by blood stem cells, largely due to the lack of tools to track these cells in humans. Our project proposed to overcome this research bottleneck by transplanting human blood stem cells into a mouse and genetically engineering these cells to develop into human iNKT cells. This “humanized” mouse model allowed us to directly follow the differentiation of human blood stem cells into iNKT cells in a living animal. At the conclusion of this project, we have proven the “TCR-instruction” model that governs the differentiation of human blood stem cells into iNKT cells. This study enriches our knowledge about human stem cells, and paves the way for developing stem cell-based iNKT cell therapies.