<p dir="ltr">Hematopoietic stem cells (HSCs) originating from aorta-gonad-mesonephros (AGM) could self-renew and develop into various immune cells, such as T cells, neutrophil and natural killer (NK) cells, rendering them as a promising cell source for immunotherapy. NK cells belong to the family of the innate lymphoid cells, and are employed as one of immunotherapy to cure solid and hematological malignancies including leukemia. Neutrophils are one of the granulocytes, and they are emerging as a new therapeutic target in various cancers. Due to the lack of reliable sources for the amounts of HSCs and immune cells required for clinical infusions (~10⁹ cells/patient), it remains as a major challenge to realize their full potential in targeted cell and immunotherapy. While substantial efforts have been made to generate native cell-like HSPCs and immune cells from human pluripotent stem cells (hPSCs), intricate molecular process governing the differentiation of HSCs and immune cells remain elusive, preventing the development of robust strategies for HSC and immune cell productions.</p><p dir="ltr">In this study, we first demonstrated that critical role of temporally regulating Wnt signaling in initiating AGM-like hematopoiesis across 11 hPSC lines. By inhibiting TGFβ at the stage of aorta-like CD34+SOX17⁺ hemogenic endothelium, which led to the downregulation of Wnt signaling, we established a chemically defined, feeder-free culture system that efficiently produced robust AGM-like hematopoietic cells. Furthermore, we investigated how hypoxia affects the <i>in vitro</i> hPSC differentiation into HSPCs, which resulted in a hypoxia-enhanced HSPC differentiation platform.</p><p dir="ltr">Next, the temporal roles of transcription factors (TFs), including <i>NFIL3</i>, <i>ID2</i>,<i> </i>and <i>SPI1</i>, in regulating and promoting NK cell differentiation from hPSCs are determined. <i>NFIL3</i> and <i>SPI1</i> have been reported to influence the early stages of NK cell development, while <i>ID2</i> has an impact on the generation of NK cells throughout the early and intermediate stage. We genetically modified hPSCs with doxycycline-inducible expression of <i>NFIL3</i>, <i>ID2</i>,<i> </i>and <i>SPI1</i>, and investigated their roles in NK cell induction from hPSCs. Among these three TFs, forced expression of <i>ID2</i> yielded the highest percentage of NK cells under a chemically defined, feeder-free monolayer culture condition, demonstrating that forced expression of NK-specific TFs improves the efficiency of NK cell differentiation from hPSCs.</p><p dir="ltr">Chimeric antigen receptor (CAR) is an artificial cell receptor expressed on immune T or NK cells that has been engineered to allow T or NK cells to re-target cancer cells by exclusively binding to a cancer-specific protein. CAR engineering has significantly improved the anti-tumor efficacy of NK cell therapy, resulting in 6 FDA-approved CAR-T therapies and many other ongoing clinical trials. Recently, a chlorotoxin (CLTX)-based CAR was developed and shown to specifically bind to a variety of heterogenous glioblastoma (GBM) cell lines. To test whether CLTX-CAR could improve the anti-tumor cytotoxicity of hPSC-derived NK cells, hPSCs were engineered with CLTX-CAR for stable and homogenous CAR expression via Cas9-mediated homologous recombination. The expression of CLTX-CAR did not affect the pluripotency and NK cell differentiation potential of hPSCs, and CLTX-CAR significantly improved the cytotoxicity of hPSC-derived NK cells against GBM cells.</p><p dir="ltr">Finally, we implemented a GBM-on-a-chip microfluidic model to interrogate the tumor microenvironment (TME). Microfluidics are an emerging device for investigating cancer biology with spatiotemporal control over signaling modulators by using a small volume. The interaction between hPSC-drived neutrophils and GBM was explored in this microfluidic device. GBM TME is very complex and involves many cell types, including neurons, microglia, immune T and NK cells. In the future, microfluidic models with isogenic cell components will be designed and implemented to better model GBM TME.</p><p dir="ltr">In summary, these discoveries confirm the pivotal role of Wnt signaling in guiding hPSCs towards hematopoietic lineages, while also highlighting <i>ID2</i> as a potent enhancer of NK cell differentiation from hPSC-derived hematopoietic progenitor cells. Additionally, CAR engineering enhances the anti-tumor capabilities of hPSC-derived NK cells. Furthermore, microfluidic models are employed to interrogate GBM TME.</p>