BACKGROUND: The CXCR4 receptor antagonist plerixafor (AMD3100) is raising interest as an anti-cancer agent that disrupts the CXCL12-CXCR4 chemokine – receptor interaction between neoplastic cells and their microenvironment in tumor progression and metastasis. Here, we investigated plerixafor for anti-cancer activity in Ewing sarcoma, a rare and aggressive cancer of bone and soft tissues. METHODS: We used a variety of methods such as cell viability and migration assays, flow cytometry, phosphotyrosine arrays and western blotting to determine plerixafor effects on five characterized Ewing sarcoma cell lines and a low-passage culture in vitro. RESULTS: Unexpectedly, plerixafor led to an increase in cell viability and proliferation in standard cell growth conditions, and to chemotactic migration towards plerixafor. Exploring potential molecular mechanisms underlying this effect, we found that Ewing sarcoma cell lines divided into classes of high- and low-level CXCR4 surface expression. Proliferative plerixafor responses were observed in both groups, maintained despite significant CXCR4 down-regulation or inhibition of Gαi-protein signal transduction, and involved activation of multiple receptor tyrosine kinases (DDR2, MERTK, MST1R, NTRK1, RET), the most prominent being platelet-derived growth factor receptor beta (PDGFRB). PDGFRB was activated in response to inhibition of the CXCL12-CXCR4 axis by plerixafor and/or pertussis toxin (Gαi-protein inhibitor). Dasatinib, a multi-kinase inhibitor of both PDGFRB and the CXCR4 downstream kinase SRC, counteracted this activation in some but not all cell lines. CONCLUSION: These data suggest a feedback interaction between the CXCR4 chemokine receptor and RTK signaling cascades that elicits compensatory cell survival signaling and can shift the net effect of plerixafor towards proliferation. PDGFRB was identified as a candidate driver RTK and potential therapeutic co-target for CXCR4 in Ewing sarcoma. Although as yet limited to in vitro studies, these findings call for further investigation in the cancer – microenvironment interplay in vivo.