p38 MAPK and nuclear factor-κB (NF-κB) signaling pathways have been implicated in the control of skeletal myogenesis. However, although p38 is recognized as a potent activator of myoblast differentiation, the role of NF-κB remains controversial. Here, we show that p38 is activated only in differentiating myocytes, whereas NF-κB activity is present both in proliferation and differentiation stages. NF-κB activation was found to be dependent on p38 activity during differentiation, being NF-κB an effector of p38, thus providing a novel mechanism for the promyogenic effect of p38. Activation of p38 in C2C12 cells induced the activity of NF-κB, in a dual way: first, by reducing IκBα levels and inducing NF-κB-DNA binding activity and, second, by potentiating the transactivating activity of p65-NF-κB. Finally, we show that interleukin (IL)-6 expression is induced in C2C12 differentiating myoblasts, in a p38- and NF-κB-dependent manner. Interference of IL-6 mRNA reduced, whereas its overexpression increased, the extent of myogenic differentiation; moreover, addition of IL-6 was able to rescue significantly the negative effect of NF-κB inhibition on this process. This study provides the first evidence of a crosstalk between p38 MAPK and NF-κB signaling pathways during myogenesis, with IL-6 being one of the effectors of this promyogenic mechanism.