Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of HIFα and the arylhydrocarbon receptor nuclear translocator(ARNT/HIF1β). Previously, we have reported that ARNT function is required for murine placental development. Here, we used cultured trophoblast stem (TS)cells to investigate the molecular basis of this requirement. In vitro, wild-type TS cell differentiation is largely restricted to spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS cells differentiated into chorionic trophoblasts and syncytiotrophoblasts, as demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the HIV receptor CXCR4. During this process, a region of the differentiating Arnt-null TS cells underwent granzyme B-mediated apoptosis,suggesting a role for this pathway in murine syncytiotrophoblast turnover. Surprisingly, HIF1α and HIF2α were induced during TS cell differentiation in 20% O₂; additionally, pVHL levels were modulated during the same time period. These results suggest that oxygen-independent HIF functions are crucial to this differentiation process. As histone deacetylase(HDAC) activity has been linked to HIF-dependent gene expression, we investigated whether ARNT deficiency affects this epigenetic regulator. Interestingly, Arnt-null TS cells had reduced HDAC activity,increased global histone acetylation, and altered class II HDAC subcellular localization. In wild-type TS cells, inhibition of HDAC activity recapitulated the Arnt-null phenotype, suggesting that crosstalk between the HIFs and the HDACs is required for normal trophoblast differentiation. Thus, the HIFs play important roles in modulating the developmental plasticity of stem cells by integrating physiological, transcriptional and epigenetic inputs.