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Summary: Extended/expanded pluripotent stem (EPS) cells can efficiently contribute to both embryonic and extraembryonic lineages in vitro and in vivo. Starting from these cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis.For complete details on the use and execution of this protocol, please refer to Li et al. (2019).