The zygote and blastomeres of cleavage stage mouse embryos have the capacity to differentiate to both embryonic and extra-embryonic lineages and are considered functionally totipotent. Until now, it has not been possible to establish stable cell lines that resemble these totipotent-like cells. Recently, we demonstrated that by modulating signalling pathways known to be important in early embryonic development it is possible to capture in vitro a self-renewing state that possessed features of preimplantation blastomeres. We reported that expanded potential stem cells (EPSCs) can be established from 8-cell (8C) embryos, individual 8-cell blastomeres, by direct conversion of mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs). Herein, we present a detailed protocol for the derivation and expansion of EPSCs in our novel media formulation, expanded potential stem cell medium (EPSCM). It is envisioned that EPSCM and EPSCs will provide a useful resource to study the earliest stages of embryonic development and lineage commitment.