Simple SummaryRhabdomyosarcoma is the most common soft tissue cancer in children and adolescents. Its resemblance to skeletal muscle tissue distinguishes rhabdomyosarcomas from other types of soft tissue cancer. The development and integrity of healthy skeletal muscle depend on a strictly regulated, hierarchically organized machinery in cells. In rhabdomyosarcoma, this process goes awry, resulting in aberrant, malignant skeletal muscle states. These aberrant skeletal muscle states define subtypes of rhabdomyosarcomas. In this review, we describe normal muscle development and summarize recent insights into how changes in rhabdomyosarcoma cells disrupt normal skeletal muscle homeostasis, thereby defining the cancerous nature of this disease. We also describe differences in myogenic differentiation characteristics between different groups of cells within the tumors. Such differences appear to be dynamic and influence the behavior of the cells. We believe that interactions between cancer genes/proteins and basic muscle programs are key to understanding the cancerous identity of rhabdomyosarcoma and may provide windows of opportunity with regard to rhabdomyosarcoma treatment.AbstractRhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children and adolescents, represents an aberrant form of skeletal muscle differentiation. Both skeletal muscle development, as well as regeneration of adult skeletal muscle are governed by members of the myogenic family of regulatory transcription factors (MRFs), which are deployed in a highly controlled, multi-step, bidirectional process. Many aspects of this complex process are deregulated in RMS and contribute to tumorigenesis. Interconnected loops of super-enhancers, called core regulatory circuitries (CRCs), define aberrant muscle differentiation in RMS cells. The transcriptional regulation of MRF expression/activity takes a central role in the CRCs active in skeletal muscle and RMS. In PAX3::FOXO1 fusion-positive (PF+) RMS, CRCs maintain expression of the disease-driving fusion oncogene. Recent single-cell studies have revealed hierarchically organized subsets of cells within the RMS cell pool, which recapitulate developmental myogenesis and appear to drive malignancy. There is a large interest in exploiting the causes of aberrant muscle development in RMS to allow for terminal differentiation as a therapeutic strategy, for example, by interrupting MEK/ERK signaling or by interfering with the epigenetic machinery controlling CRCs. In this review, we provide an overview of the genetic and epigenetic framework of abnormal muscle differentiation in RMS, as it provides insights into fundamental mechanisms of RMS malignancy, its remarkable phenotypic diversity and, ultimately, opportunities for therapeutic intervention.