Mouse embryonic stem cells (ESCs) have great potential to understand fundamental biological processes and to act as a potential source of stem cells therapy, informing advances in regenerative medicine and treatments for various illnesses. The preimplantation genetic diagnosis (PGD) technique, involving embryonic cell biopsy while ensuring embryo viability was employed in the establishment of ESCs. In this research, all eight single blastomeres were derived from 8-cell embryos utilizing PGD-like technique with assistance of XYClone laser and subsequently cultured in basic stem cell (SC) medium. As depicted in the results, single blastomeres isolated 8-cell mouse embryos were able to develop to the ESCs clusters on day 7 post-biopsy and exhibited similar morphology to ESCs. Despite morphological variations on day 3, including clusters and blastocoel, the duration for single blastomeres to form ESCs clusters on day 7 in SC medium remained similar. The ability of single blastomeres to form clusters (39.6% ± 1.7%) was greater than that of blastocoels (35.7% ± 1.9%) on day 3 after culture. By day 7, both groups had formed ESCs clusters, of which those that formed blastocoels and clusters on day 3 had ESCs cluster development rates of 17.4% ± 0.4% and 25.5% ± 2.3%, respectively. Furthermore, these clusters of ESCs derived from single blastomeres are capable of expanding over time. In conclusion, single blastomeres collected from the 8-cell mouse embryos could develop into ESCs clusters when cultured in basic SC medium and maintained after further culture.