The p53 oncosuppressor protein regulates cell cycle checkpoints and apoptosis, but increasing evidence also indicates its involvement in differentiation and development. We had previously demonstrated that in the presence of differentiation-promoting stimuli, p53-defective myoblasts exit from the cell cycle but do not differentiate into myocytes and myotubes. To identify the pathways through which p53 contributes to skeletal muscle differentiation, we have analyzed the expression of a series of genes regulated during myogenesis in parental and dominant-negative p53 (dnp53)-expressing C2C12 myoblasts. We found that in dnp53-expressing C2C12 cells, as well as in p53(-/-) primary myoblasts, pRb is hypophosphorylated and proliferation stops. However, these cells do not upregulate pRb and have reduced MyoD activity. The transduction of exogenous TP53 or Rb genes in p53-defective myoblasts rescues MyoD activity and differentiation potential. Additionally, in vivo studies on the Rb promoter demonstrate that p53 regulates the Rb gene expression at transcriptional level through a p53-binding site. Therefore, here we show that p53 regulates myoblast differentiation by means of pRb without affecting its cell cycle-related functions.