The nonphysiological placental oxidative environment has been implicated in many complications during human pregnancy. Oxygen tension can influence a broad spectrum of molecular changes leading to alterations in trophoblast cell lineage development. In this study, we report that mouse wild-type trophoblast stem cells (TSCs) react to low oxygen (3%) with an enhanced differentiation into the giant cell pathway, indicated by a downregulation of the early stem cell markers Eomes and Cdx2 as well as by a significant upregulation of Tfap2c and the differentiation markers Tpbpa and Prl3d1. Here we demonstrated that connexin 31/GJB3-deficient TSCs failed to stabilize HIF-1A under low oxygen, resulting in nonresponsiveness of different marker genes, such as Cdx2 and Eomes and Tfap2c and Tpbpa. Moreover, connexin 31-deficient TSCs revealed a shift in giant cell differentiation from Prl3d1 expressing parietal giant cells to Ctsq, Prl3b1, and Prl2c2-positive giant cells, probably sinusoidal and canal lining trophoblast giant cells. Thus, loss of connexin 31 led to different giant cell subtypes which bypass the progenitor regulators Tfap2c and Tpbpa under low oxygen conditions.