Multipotent trophoblasts undergo dynamic morphological movement and cellular differentiation after conceptus implantation to generate placenta. However, the mechanism controlling trophoblast development and differentiation during peri-implantation development in human remains elusive. In this study, we modeled human conceptus peri-implantation development from blastocyst to early postimplantation stages by using an in vitro coculture system and profiled the transcriptome of 476 individual trophoblast cells from these conceptuses. We revealed the genetic networks regulating peri-implantation trophoblast development. While determining when trophoblast differentiation happens, our bioinformatic analysis identified T-box transcription factor 3 (TBX3) as a key regulator for the differentiation of cytotrophoblast (CT) into syncytiotrophoblast (ST). The function of TBX3 in trophoblast differentiation is then validated by a loss-of-function experiment. In conclusion, our results provided a valuable resource to study the regulation of trophoblasts development and differentiation during human peri-implantation development.