The leukemia inhibitory factor (LIF)/glycoprotein 130 (gp130)/signal transducer and activator of transcription 3 (STAT3) pathway plays an essential role in the maintenance of self-renewal and pluripotency in mouse embryonic stem (ES) cells. However, in primate ES cells, including those from humans and monkeys, LIF alone is not sufficient to maintain self-renewal. The precise role of the LIF/gp130/STAT3 pathway for self-renewal in primate ES cells is still unclear. In this study, we found that stimulation of cynomolgus monkey ES cells with LIF or interleukin (IL)-6/soluble IL-6 receptor leads to STAT3 phosphorylation, an effect seen previously in murine ES cells. Concomitant with this notion, nuclear translocalization and transcriptional activation of STAT3 were observed in a LIF-dependent manner. Moreover, the analysis of a dominant interfering mutant, STAT3F, showed that even though the phosphorylation, nuclear translocalization, and transcriptional activation of endogenous STAT3 after LIF stimulation were completely abrogated by over-expressing STAT3F in monkey ES cells, they continued to proliferate in an undifferentiated state, retaining their pluripotency. These results demonstrate that the LIF/gp130/STAT3 pathway functions in cynomolgus monkey ES cells but is not essential for the maintenance of self-renewal. They also suggest that cynomolgus monkey ES cells, unlike murine ES cells, are maintained in an undifferentiated state through LIF/gp130/STAT3-independent signaling.