Salamanders display unique regeneration abilities among adult vertebrates. An intriguing feature of salamander regeneration is the dedifferentiation of cells, such as myofibers and myotubes at the injury site, a process that involves cell cycle reentry from the differentiated state. A thrombin-activated serum factor that is distinct from conventional growth factors is known to cause S-phase reentry in salamander myotubes. While mammalian myotubes do not reenter S-phase upon serum stimulation, an upregulation of some immediate early genes such as jun and fos has been observed. Until now, it was unknown whether this transcriptional response was stimulated by conventional growth factors or by the thrombin-activated serum factor. By measuring transcriptional activity in individually purified C2C12 mouse myotubes using quantitative reverse transcription polymerase chain reactions, we show that a set of immediate early genes are activated in response to the thrombin-activated serum factor in a distinct manner from the growth factors PDGF, FGF and EGF. A partially purified fraction of the thrombin activated serum factor elicited stronger upregulation of a broader set of genes compared to individual growth factors and additionally caused downregulation of E2F6. Despite this robust transcriptional response in mammalian myotubes, we did not detect a large-scale change in histone H3K9 di-methylation or S-phase, a feature that characterizes salamander serum-stimulated myotubes. Our results indicate that mammalian myotubes have retained responsiveness to the thrombin-activated serum factor, but full reentry into S-phase is prevented by factors downstream of the immediate early genes.