Rhabdomyosarcoma (RMS) is the most common malignant soft-tissue tumor of childhood. Nearly 15% of children present with metastatic disease, frequently involving the lungs and bone marrow. The prognosis for patients with metastatic RMS is dismal, with an estimated 3-year overall survival of 30%. Stromal-cell derived factor 1-alpha (SDF1alpha, CXCL12) is a chemokine that plays a crucial role in the metastatic attraction of tumor cells expressing its receptor, CXCR4. We investigated the role of the bone marrow microenvironment on RMS signaling through the CXCR4/SDF1alpha pathway in cell lines and primary tumors. Conditioned media (CM) isolated from cultured patient-derived bone marrow stromal cells (BMS) induced migration and proliferation in multiple RMS cell lines. CXCR4 was expressed in RMS cell lines and primary tumors, with higher expression in alveolar subtype RMS. Further, SDF1alpha was secreted by all BMS cultures and potently induced the migration and proliferation of RMS cells. Small molecule or blocking antibody-mediated inhibition of CXCR4 or SDF1alpha suppressed RMS cell migration towards BMS-CM, confirming the activity of this axis. Our study provides strong evidence for the involvement of the bone marrow microenvironment and CXCR4/SDF1alpha signaling in metastasis of RMS. These results form the basis for future studies to delineate the mechanisms of bone marrow metastasis in RMS.