Rapidly proliferating pre-implantation embryos and their derived cells, called as embryonic stem cells (ESCs) are vulnerable to various genotoxic insults. Any unrepaired damage at this critical stage can give rise to either miscarriage or later prenatal abnormalities. However, the mechanisms through which the ESCs and early-stage embryos respond to genotoxic stress is not fully studied. Through in vivo chimera assay, we have recently shown that replication stress (RS)-induced ESCs contribute to the extra-embryonic compartment of the embryos in contrast to the intact cells which contribute only to the embryonic proper. In vivo chimera assay possesses various limitations including technical and ethical challenges. Here, to overcome these limitations and to uncover the molecular mechanisms underlying our previous findings, we studied the impact of RS on in vitro differentiation of ESCs to the extra-embryonic lineages. Briefly, we found that RS increases the expression of key trophectoderm (TE) markers in ESCs. Moreover, under trophoblast stem cells (TSCs) culture condition, we demonstrated that RS induces the differentiation of ESCs toward TSCs and increases the number of trophoblast-like stem cells (TLSCs). Next, to understand whether the ESC-derived TSCs (i.e., TLSCs) are functionally similar to the embryo-derived TSCs, we established an innovative combinatorial approach of ESC in vitro differentiation with 3D stem cell embryogenesis. Concisely, we could generate structures from the assembly of TLSCs and ESCs. Interestingly we found that these structures are characteristically similar to the structures generated from ESCs and embryo- derived TSCs. Through such approach we studied the impact of RS on the differentiation potential of ESCs. Strikingly, we found that RS-induced TLSCs are able to give rise to the higher number of embryo-like structures. To further consolidate these findings, we studied the impact of RS on the terminal differentiation of ESCs toward trophoblast giant cells (TGCs). Surprisingly, we found that RS induces the differentiation of ESCs toward TGCs and increases the number of trophoblast-like giant cells (TLGCs). Finally, we focused on the molecular mechanisms through which RS induces the differentiation of ESCs toward TSCs. In brief, we found the increased expression of TE genes in ESCs is mediated through ATR-Chk1 pathway and does not require the activation of ATM-Chk2 pathway. We also showed that RS increases the expression of Tead4, the master regulator of TE-specific transcriptional program. We demonstrated that the expression of Tead4 in RS-induced ESCs is not ATR-Chk1-dependent however, its binding to the transcription factor binding motifs (TFBMs) of TE genes is mediated through ATR. Ultimately, we found that the ATR-Chk1-mediated binding of Eomes to its TFBM further enhances the expression of this TE-specific gene in RS-induced ESCs. Overall, these findings reveal a role of ATR-Chk1-dependent transcriptional regulation of TE genes in inducing the ESCs differentiation toward TSCs in response to RS.