<p>In children, rhabdomyosarcoma is the most common tumor originating in soft connective tissue. The PAX3/PAX7-FOXO1 fusion-positive alveolar subtype has poor clinical outcomes, with frequent recurrence, metastasis, and low survival. Current therapies may be limited by the tumor microenvironment containing cancer, immune, and fibroblast cells. In cancer, cells that transform into cancer-associated fibroblasts support growth and other malignant properties of the tumor. To study the role of cancer-associated fibroblasts, we isolated cells from the lung metastases of a patient with rhabdomyosarcoma that are positive for fibroblast biomarkers. Compared to normal lung fibroblasts, these cancer-associated fibroblasts secreted distinct factors that specifically supported the growth and migration of fusion-positive rhabdomyosarcoma cells. The cancer-associated fibroblasts also promoted expression of immune checkpoints and conferred resistance to cyclophosphamide, a chemotherapy commonly used to treat this disease. We further characterized the secretory phenotype of cytokines and growth factors produced by these cancer-associated fibroblasts and targeted CXCR4 expression inhibition, which induced cytotoxicity at increased sensitivity. This study establishes a model of cancer-associated fibroblasts from metastatic fusion-positive rhabdomyosarcoma. Altogether, our results describe tumor-promoting mechanisms of growth, migration, and treatment resistance supported by the tumor microenvironment, and offer a novel therapeutic strategy for the treatment of rhabdomyosarcoma.</p>