Myf5 plays a central role in determination of the myogenic lineage, yet the signalling pathways that control its activation remain unclear. In adult muscle, Myf5 is expressed in satellite cells and muscle spindles but not by myonuclei. However, Myf5 expression is activated in myonuclei in response to muscle denervation. This can be modelled in culture using Myf5nlacZ/+ mice, allowing signalling pathways controlling Myf5 to be readily examined. We found that mitogen-rich medium induces activation of the Myf5 locus through calcium, which interacts with calmodulin to promote calcineurin and calmodulin kinase. Calcineurin activates NFAT to control Myf5 activation, while p38/JNK activity prevents activation by this route. Calmodulin kinase however, acts predominately through ERK signalling to activate Myf5. Interestingly, we found that IGF-1 can substitute for mitogen-rich medium and activates Myf5 through calcium, PI3K and ERK pathways. Together these observations show that Myf5 activation in adult muscle is accomplished by a complex signalling pathway, and provides candidates that can be examined for their role in Myf5 regulation during development.