Background <p>The characteristic constituents of the mature placenta are made up of highly specialised trophoblast cell types. Trophoblast stem cells (TSCs) possess the developmental plasticity to differentiate into all these mature placental cell types. However, TSCs are typically a heterogenous population with individual cells exhibiting varying degrees of stem cell marker expression. Moreover, standard differentiation protocols of mouse TSCs are based on a release from the stem cell state and result in a mixed population of various trophoblast cell types. This mix of differentiating cells is a particular impediment for functional investigations into the roles of specific trophoblast subtypes of the mouse placental labyrinth, i.e. the portion of the placenta that establishes the feto-maternal exchange unit.</p> Methods <p>Murine TSCs were used to establish culture protocols that enhance the stem cell state of mouse TSCs and that drive differentiation into specific labyrinth trophoblast cell types. TSCs were treated with a panel of 35 epigenetic inhibitors and with 12 selected small molecule compounds either alone or in combination, and across a wide range of doses. TSC stemness and differentiation was assessed by RT-qPCR to determine the relative expression of trophoblast cell type-specific marker genes, and by immunofluorescence staining to verify enrichment of the cell type(s) of interest.</p> Results <p>TSC markers CDX2 and SOX2 were robustly enriched upon treatment of mouse TSCs with the KDM1A inhibitor GSK-LSD1, indicative of an enhanced stem cell state. Treatment of differentiating TSCs with the LIMK2 inhibitor BMS-3 in conjunction with either the PPARG agonist rosiglitazone (Rosi) or with Tunicamycin and GSK-LSD1 promoted differentiation of labyrinth trophoblast cell types in general, at the expense of junctional zone trophoblast. Rosi in combination with the KDM1A1 inhibitor GSK-LSD1 or BMS-3 enriched for syncytiotrophoblast layer I cells and sinusoidal trophoblast giant cells, while high doses of Rosi resulted specifically in sinusoidal trophoblast giant cell differentiation. Rosi in combination with the protein synthesis inhibitor Tunicamycin enriched for syncytiotrophoblast layer I cells only. Activin A and the WNT agonist Chiron99021 resulted in predominant syncytiotrophoblast layer II differentiation.</p> Conclusion <p>Collectively, we establish a roadmap of treatment regimens that promote the differentiation of mouse TSCs into specific trophoblast cell types of the feto-maternal exchange surface. These insights will enable refined biochemical and molecular assessment strategies on defined trophoblast cell types that govern reproductive outcome.</p>