This thesis comprises two parts, each contributing novel insights into self-organized patterning and differentiation in human pluripotent stem cells (hPSCs). Self-organized patterning of mammalian pluripotent stem cells on micropatterned surfaces has been established as an in vitro platform for early developmental studies, complementary to in vivo animal models. The first project addressed the limitations of current micropatterning techniques, due to complex fabrication processes, such as micro-contact printing, preventing widespread usage in biological research. We developed a projection stereolithography-based micropatterning method that uses a digitally tunable photomask to rapidly print hydrogel with micro-features onto glass-bottomed-culture vessels. Combined with the laminin-521 (or LN521) extracellular matrix coating, this technology provides a surface suitable for hPSC attachment and growth with minimal non-specific cell adhesion. The study demonstrated the self-patterning results of hPSCs following gastrulation and ectodermal induction produced on our micropatterned surface are comparable with those obtained using commercially available micropatterned plates. This novel micropatterning approach enables customizable, rapid fabrication of micropatterned surfaces for cell study at a reduced cost, with potential application in developmental biology and regenerative medicine research. The second project explores cadherin switching during the epithelial-mesenchymal transition (EMT) in the differentiation trajectory of hPSCs through the primitive streaks (PS) and into mesodendermal subtypes. We measured EMT, and cadherin switching (E-cadherin downregulation and N-cadherin upregulation) during hPSC differentiation to PS and subsequently to distinct mesendodermal subtypes using established protocols and variants in signaling modulation of the key pathways, i.e., Activin, BMP, and Wnt. The findings reveal that while early signaling perturbations largely affected the extent of cadherin switching, the differentiation potential of PS cells was unimpacted. Specifically, definitive endoderm progenitors retained the ability to differentiate into both endodermal and mesodermal fates, while PS cells in mid to posterior regions exhibited restricted potential toward definitive endoderm. Additionally, E-Cadherin knockout did not alter cell fate outcomes in mesendodermal differentiation. Overall, the project revealed the decoupling of cadherin dynamics from cell fate decisions in mesendodermal differentiation through PS coordinates, with translational potential for cancer and age-related degenerative disease studies, where modulating EMT and cadherin switching could support innovative therapy development.