Rhabdomyosarcoma is the most commonly diagnosed pediatric soft tissue carcinoma and involves cells in the skeletal muscle lineage. The disease is classified into two main subtypes: embryonal rhabdomyosarcoma (ERMS) which is associated with a more favorable clinical outcome, and the less common alveolar rhabdomyosarcoma (ARMS) which has higher rates of metastasis, drug resistance, recurrence, mortality, and an overall poorer prognosis. Most ARMS tumors are characterized by a t(2;13)(q35;q14) chromosomal translocation that produces a fusion protein from the embryogenesis-associated gene PAX3 and FOXO1a. The chimeric protein PAX3-FOXO1a acts as a transcription factor capable of inhibiting myoblast differentiation, however, the complexity of myogenesis suggests multigenic regulation. The present goal is to determine the role of miRNAs in the etiology of rhabdomyosarcoma and the regulation of myogenesis by taking a more comprehensive approach. In this study, we use RNA-sequencing-based technology to assess the miRNA expression profiles of rhabdomyosarcoma cell lines (RD and Rh30) that are either positive or negative for the PAX3-FOXO1a fusion protein, and compare the profiles to those of undifferentiated and differentiated primary skeletal muscle cells. Additionally, we define the impact PAX3-FOXO1a has on expression of specific miRNAs by modulating fusion protein levels in tumor cell lines