Self-renewal of in vitro cultured mouse embryonic stem (mES) cells is dependent on the presence of leukemia inhibitory factor (LIF). LIF induces overexpression and tyrosine phosphorylation of STAT3 (signal transducer and activator of transcription 3) and its subsequent nuclear translocation. The molecular chaperone heat shock protein 90 (Hsp90) is involved in the activation and maturation of a wide variety of substrate proteins. We investigated the effect of LIF withdrawal on the protein expression levels of STAT3 and Hsp90 and on the interactions between STAT3 and Hsp90. Taken together the data presented here suggest that LIF promotes the interaction of Hsp90 with STAT3 during self-renewal, indicating a potentially pivotal role for Hsp90 in the LIF-based maintenance of self-renewal of mouse embryonic stem cells.