Mouse ES (embryonic stem) cells are maintained in an undifferentiated state in the presence of LIF (leukaemia-inhibitory factor). In general, LIF engages a heterodimeric receptor complex composed of a low-affinity LIF receptor (LIFRβ) and gp130, and activates STAT3 (signal transducers and activators of transcription 3) and ERKs (extracellular signal-regulated kinases). However, in undifferentiated ES cells in the presence of LIF, STAT3 is phosphorylated but ERKs are not. The removal of LIF-induced dephosphorylation of phospho-STAT3 and phosphorylation of ERKs resulted in the differentiation of ES cells. Here, we show that the dephosphorylation of phospho-STAT3 corresponds to the activation of ERKs pathway from the time-courses of the phosphorylation levels in detail. We found that the treatment of membrane-permeable STAT3IP (STAT3 inhibitory peptide), which inhibits homodimeric formation of STAT3, induced the phosphorylation of ERKs in ES cells in the presence of LIF. In addition, the removal of LIF decreased the expression level of SOCS3 (suppressor of cytokine signalling 3), a negative regulator of LIF signalling, and the phosphorylation of ERKs was efficiently induced in the ES cells where SOCS3 was down-regulated. These results suggested that LIF-induced SOCS3 suppressed the ERKs activation pathway in undifferentiated ES cells, and the down-regulation of SOCS3 by the removal of LIF triggered the phosphorylation of ERKs.