Nuclear transfer can be used to generate embryonic stem cell lines from somatic cells, and these have great potential in regenerative medicine. However, it is still unclear whether any individual or cell type can be used to generate such lines. Here, we tested seven different male and female mouse genotypes and three cell types as sources of nuclei to determine the efficiency of establishing nuclear transfer embryonic stem cell lines. Lines were successfully established from all sources. Cumulus cell nuclei from F(1) mouse genotypes showed a significantly higher cumulative establishment rate from reconstructed oocytes than from other cells; however, there were no genotype differences in success rates from cloned blastocysts. Thus, the overall success depends on preimplantation development, and, once embryos have reached the blastocyst stage, the genotype differences disappear. All mouse genotypes that were tested demonstrated at least one cell line that subsequently contributed to germline transmission in chimeric mice, so these cell lines clearly possess the same potential as embryonic stem cells derived from fertilized embryos. Thus, nuclear transfer embryonic stem cells can be generated relatively easily from a variety of inbred mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly.