AbstractThe pluripotent stem cells exist in a narrow window during early development and its derivation depends on intrinsic and extrinsic growth signalling in vitro. It has remained challenging to derive two or three distinct cell lines that are representative of blastocyst‐stage lineages from one preimplantation embryo simultaneously in a chemical defined condition. Therefore, it is desirable to establish a system by manipulating extrinsic signalling in culture to derive multiple types of stem cells from a single blastocyst. This study used a defined medium containing Activin A, WNT activator and LIF (ACL medium), enabling establishment of ACL‐ESCs and ACL‐XEN cells from one blastocyst. ACL‐blastoids were generated by suspending ACL‐ESCs and ACL‐XEN cells with ACL‐blastoid medium in three‐dimensional culture system. Lineage markers expression of ACL‐blastoids were performed by immunofluorescence. Our results indicate that ACL‐ESCs and ACL‐XEN cells derived from one blastocyst represent ICM and PrE lineages. Importantly, we obtained ACL‐blastoid from ACL‐ESCs and ACL‐XEN cells self‐aggregation, partially recapitulating early development and initiation of early implantation events. This study would not only provide ACL culture system for derivation and maintenance of two types of cell lines corresponding to ICM as well as PrE, but also reconstruct blastoids with them to deepen our understanding of early embryogenesis and widen insights into translational application of stem cells.