Trophectoderm (TE) and the inner cell mass are the first two lineages in murine embryogenesis and cannot naturally transit to each other. The barriers between them are unclear and fascinating. Embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) retain the identities of inner cell mass and TE, respectively, and, thus, are ideal platforms to investigate these lineages in vitro. Here, we develop a loss-of-function genetic screening in haploid ESCs and reveal many mutations involved in the conversion of TSCs. The disruption of either Catip or Dyrk1a (candidates) in ESCs facilitates the conversion of TSCs. According to transcriptome analysis, we find that the repression of Dyrk1a activates totipotency, which is a possible reason for TE specification. Dyrk1a-null ESCs can contribute to embryonic and extraembryonic tissues in chimeras and can efficiently form blastocyst-like structures, indicating their totipotent developmental abilities. These findings provide insights into the mechanisms underlying cell fate alternation in embryogenesis.