We have determined the frequency at which embryonic stem cell (ESC) lines can be established from inner cell masses (ICMs) isolated from blastocysts homozygous for lethal mutations in the mouse t-complex. Approximately one-third of the expected number, 3/29, of the ESC lines established from embryos obtained by inter-se mating of +/tw18 mice are homozygous for the tw18 haplotype. These tw18/tw18 ESC lines form a variety of cell types in vitro and in vivo, including mesodermal derivatives such as cartilage and muscle. On the basis of these and data from other studies, we suggest that the normal function of the gene represented by the tw18 lethal allele is required for multiplication/survival of mesodermal precursors in the embryo rather than the specification of the mesodermal lineage, and that the lethal effects of this mutation are expressed in only the highly structured environment of the early postimplantation embryo. In studies of the lethal tw5 haplotype, we found that 2/2 ESC lines obtained are mutant homozygotes. Analysis of these data, in conjunction with the results of our earlier study (Magnuson, T., Epstein, C. J., Silver, L. M., and Martin, G. R. (1982), Nature (London) 298, 750-753), suggests that homozygosity for the genes found in the tw5 haplotype does not reduce cell viability. By contrast, 0/16 ESC lines isolated from embryos obtained from matings of +/t0 mice are mutant homozygotes. Analysis of the genotypes of ICM-derived primary stem cell colonies suggests that t0 homozygous ICM cells are unable to undergo sufficient proliferation in vitro to give rise to ESC lines.