C57BL/6 mouse is the most standard strain in mouse genetics. The strain does, however, have several disadvantages; one being the difficulty in establishing embryonic stem (ES) cells. No reliable C57BL/6 ES cell line is widely available for creating mutant mice through gene targeting. It also greatly favors mouse genetics if one can routinely make multiple mutations by stably culturing germline-competent C57BL/6 ES cells or if one can routinely establish ES cells from C57BL/6-derived mutant mice to make multiple mutations. Recently, an ES culture method with three inhibitors (3i: SU5402 for FGFR, PD184352 for ERK, and CHIR99021 for GSK3) has been reported. Here we show that this 3i method is extremely instrumental in establishing and culturing germline-competent ES cells in the C57BL/6N strain.