Leukemia inhibitory factor (LIF) is an important regulator of skeletal muscle regeneration and has been suggested to be mitogenic for myogenic cells because it has been shown to increase the quantity of myoblast cells grown in culture over extended periods of time. Using the established C2C12 murine myoblast cell line, we observed that LIF treatment did not significantly increase the rate at which myoblasts synthesise DNA under conditions which increased cell quantity by 73% above control, whilst the known mitogen fibroblast growth factor-2 significantly increased DNA synthesis under these conditions. Consequently, we examined the capacity of LIF to prevent apoptotic cell death. LIF treatment significantly reduced staurosporine-induced apoptotic DNA fragmentation by 37% compared to control and also reduced the proteolytic activation of caspase-3 by 40% compared to control. This effect of LIF was completely abolished by addition of the phosphatidylinositol 3-kinase inhibitor wortmannin, indicating that the phosphatidylinositol 3-kinase signalling pathway, previously shown to be linked to LIF-dependent increases in cell number, is necessary in mediating the anti-apoptotic effects of LIF. LIF treatment was also associated with increased levels of Bcl-xL and XIAP transcripts compared to control. Therefore, we suggest that the role of LIF in skeletal muscle regeneration and myogenesis is that of a survival factor rather than a mitogen.