The connexin (Cx) expression during placental development in rodents is subject to exacting spatiotemporal regulation. Following implantation, Cx31 characterizes the early trophoblast cell lineage and is expressed by the spongiotrophoblast during placental development until birth. Inactivation of the Cx31 gene results in a transient placental dysmorphogenesis with an imbalance in the trophoblast cell lineage differentiation in favor to giant cells [Dev. Biol. 231 (2001) 334]. In this study, we show that trophoblast stem (TS) cells exhibit the same connexin expression found in trophoblast cell lineage differentiation. Undifferentiated TS cells exclusively express Cx31 protein and Cx31.1 transcripts. Upon differentiation of TS cells, placental-specific Cx26 and Cx43 are induced. Cx31 knockout TS cells revealed an accelerated differentiation process to giant cells compared to controls, indicated by an overall shift in expression of connexins and marker genes such as Mash2, Pl-1, and Tpbpa. Moreover, proliferation was significantly reduced in Cx31 knockout TS cells upon differentiation. Both wild type and Cx31 knockout TS cells are able to invade and erode host vessels when injected into nude mice. We conclude that during trophoblast cell lineage differentiation, the Cx31 gap junction channel is involved in maintaining the proliferative diploid trophoblast cell population.