Human rhabdomyosarcomas (RMSs) frequently demonstrate genetic alterations in ras and p53. To investigate their possible involvement in the tumorigenesis, we generated a knock-in mouse line with oncogenic K-ras, conditionally expressed by Cre/LoxP system on a background of p53 alteration. Electroporation of Cre expression vector in skeletal muscle tissues resulted in the generation of tumor in adults with tumor incidences of 100% at 10 weeks and 40% at 15 weeks, in p53−/− and p53−/+ backgrounds, respectively. The tumor histology was pleomorphic RMS with characteristic bizarre giant cells, positive for desmin and α-sarcomeric actin and exhibiting remarkable increase in total and phosphorylated extracellular signal-regulated protein kinase (ERK) 1 and ERK2. Loss of the wild-type p53 was detected in K-rasG12V-expressed tumors of p53−/+ mice. Early lesions 3 weeks after electroporation consisted of proliferating populations of myogenic progenitors, including stem cells positive for ScaI antigen, immature cells positive for desmin and neural cell adhesion molecule-positive myotubes. Thus, cooperation of oncogenic K-ras and p53 deficiency resulted in the development of pleomorphic RMS in adult mice, providing a useful mouse model for further detailed studies.