The spontaneous differentiation of mouse-derived ES cells is suppressed by the supplementation of mouse Leukemia Inhibitory Factor (mLIF) into the medium. ES cells immediately differentiate when cultured in mLIF-free medium. Although the effects of mLIF on ES cell differentiation are well known, its effects on cell proliferation have hardly been investigated. Furthermore, no report has been published on the effects of mLIF in medium on cell viability. C60 fullerene has potent antioxidant capacity but little toxicity in in vivo and in vitro studies. Therefore, the effects of C60 fullerene on the viability of ES-D3 cells were examined in three-dimensional culture with and without mLIF, demonstrating no significant difference due to the supplementation of mLIF. Thus, we obtained data required for examining the effects of mLIF on the differentiation of ES-D3 cells.