Time course of TRAP⁺ MNC formation in the osteoclast cultures after 5 days of induction culture. The culture system A of Fig 1 was applied with a modification during the osteoclast culture phase. Ten thousand D3 ES cells cultured on OP9 for 5 days were seeded on ST2 or OP9 and changed to the osteoclast culture condition for the indicated period, and TRAP⁺ MNCs were counted. A serial dose of M-CSF was added into OP9 cultures in the osteoclast culture phase. Numbers of TRAP⁺ MNCs are given as means ± SD of the triplicate cultures.