Objective To investigate the effects of developmental stages of tetraploid embryos on mouse chimeras from embryonic stem cells (ES). Methods Tetraploid embryo complementation and microinjection were utilized to produce ES mice that derived completely from ES cells. Tetraploid embryos were firstly prepared by electrofusion of 2-cell mouse embryos, and then embryonic stem (ES) cells with different genetic background (hybrid or inbred) were injected into tetraploid 1-cell, 4-cell, and blastocyst stage embryos. The injected embryos were transferred into uterine horns of pseudopregnant 2.5days female CD1 mice. The CD1 mice were subjected into cesarean sections after 16 days. Results Our data showed that 92.45% of 2-cell embryos were electrofused, 93.51% and 90.42% of the electrofused embryos developed to 4cell and blastocyst stages, respectively. Blastocysts were injected with hybrid ES cells and 22 ES mice were obtained by Cesarean section. Blastocyst was more effective than 1-cell and 4-cell. ES mice had normal germline transmission capability and the same coat color as the hybrid mice which hybrid ES cells were drived from. Conclusion The tetraploid embryo developmental stage is able to affect the ES mou