The survival probability of mouse blastocysts developed from nuclear transfer (NT) was estimated prior to embryo transfer using a novel biopsy method. NT embryos were separated into two at the 2-cell stage to produce monozygotic twin blastocysts from a single NT oocyte. We first examined efficient culture methods to produce monozygotic twin blastocysts. Then one of the two blastocysts was used for gene expression analysis, and the other was transferred to a recipient mouse. Co-culture of monozygotic twin blastomeres with fertilized embryos or culture in a small well in vitro improved the developmental potential to the blastocyst stage and increased the blastocyst cell number. Although 21.9% of twin somatic cell NT embryos had the same expression levels of Oct4 and Sox2 genes as fertilized embryos, no fetuses were obtained after transfer to recipients.