The nonobese diabetic (NOD) mouse is a valuable model for human type 1 diabetes and the development of humanized mice. Although the importance of this mouse strain is widely recognized, its usefulness is constrained by the absence of NOD embryonic stem (ES) lines with adequate germline transmission competence. In the present study, we established two germline transmission-competent types of cell lines from NOD mice; these cell lines, male germline stem (GS) cells and ES cells, were derived from NOD spermatogonia and blastocysts, respectively. NOD-GS cells proliferated in vitro and differentiated into mature sperm after transplantation into testis. NOD-ES cell lines were effectively established from NOD blastocysts using culture medium containing inhibitors for fibroblast growth receptor, MEK, and GSK3. Both the NOD-GS and NOD-ES cell lines transmitted their haplotypes to progeny, revealing a novel strategy for gene modification in a pure NOD genetic background. Our results also suggest that the establishment of GS cells is an effective procedure in nonpermissive mouse strains or other species for ES cell derivation.