In mammals, haploidy is normally restricted to the post-meiotic stages of germ line cells and represents the end of cell proliferation, which means that physiological haploidy is incompatible with self-renewal. The successful establishment of androgenetic haploid embryonic stem cell broadened the practical prospect of haploid stem cells, which means, pluripotency, self-renewal, and haploidy can be incorporated together in a single cell line. These haploid ESCs contained only the paternal set of chromosomes and shown pluripotency as well as self-renewal capabilities. Haploid embryonic stem cell had drawn great interest of researchers because of its potential in genetic modification and drug screening. Continuous technological progress in mammalian genetics depended on the availability of mouse embryonic stem cells. Some reverse genetic approaches had taken advantage of the pluripotency of genetically engineered mouse haploid embryonic stem cell to produce animals with germline-transmitted mutations. To generate androgenetic haploid mouse embryos, we performed nuclear transfer (NT) technology, in which a single sperm head from C57/BL6 × DBA/2 F1 mice, instead of a somatic nucleus, was injected into an enucleated oocyte from C57/BL6 × DBA/2 F1 mice. The androgenetic haploid embryo underwent development from 1-cell stage to blastocyst stage (Fig. 1). Among the 206 haploid embryos that were reconstructed, 30 (14.5%) developed into blastocysts in vitro. After removal of the zona pellucida, blastocysts were cultured in a standard embryonic stem cell (ESC) culture system supplemented with 2i. As the result shown, the unsatisfied blastocyst rate and blastocyst quality is still an obstacle to the derivation of androgenetic haploid stem cells. To improve the quality of androgenetic haploid blastocyst and the efficiency of the androgenetic haploid stem cells derivation, we assessed the expression of Xist gene in the androgenetic haploid embryos during the preimplantation development of mice in vitro. The result showed that the blastocyst rate of androgenetic haploid embryo was 10%﹣14% (Table 1), the time for the development to blastocyst stage varied from 3.5 day to 5.5 day. RNA-FISH results indicated that Xist gene was actively expressed in the early developmental stage, but was silenced in these embryos which developed to blastocyst stage (Fig. 2). Xist Knockdown by siRNA improved the quality of the blastocyst and the rate of outgrowth when seeded on the feeders, but did not improved the blastocyst rate. These results indicated that the expression of Xist gene is one of the factors for the unsatisfied quality of the androgenetic haploid blastocyst and the low efficency of androgentic haploid stem cell derivation.