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Background Immunotherapy is challenging in cold tumors owing to their low T-cell infiltration and prevalence of immunosuppressive myeloid cells. Chemokine gradients are critical regulators of immune cell infiltration and migration into solid tumors. As such, a comprehensive understanding of the role of chemokines in the tumor microenvironment (TME) is crucial for improving treatment strategies in cold solid tumors.Methods We employed a novel series of squamous cell carcinoma (SCC) cell lines established by our lab to identify chemokines and cytokines associated with a cold TME and validated the human relevance of our findings in a pan-cancer patient cohort spanning 12 cancer types. We identified CX3CL1 as associated with cold tumors, excising it from multiple cold SCC cell lines using CRISPR-Cas9 gene editing. CX3CL1 knockout cell lines were implanted in mice, and resulting tumors were treated with an immune checkpoint inhibitor (ICI) combination of anti-PD1 and anti-CTLA4. Treatment efficacy was evaluated by monitoring tumor growth, and immune cell populations were analyzed using flow cytometry.Results We show that elevated CX3CL1 is a feature of cold tumors. However, deletion of CX3CL1 in cold SCC cell lines was sufficient to transform the TME into an immune-hot phenotype, increasing both T and dendritic cell infiltration, suggesting CX3CL1’s dominant influence on the TME. The deletion of CX3CL1 further sensitized cold, ICI-resistant tumors to ICI treatment, delaying tumor growth and increasing infiltration of activated, tumor-specific (PD1 +CD39+) CD4 and CD8 T cells only in CX3CL1 knockout tumors. In addition to the local effects of CX3CL1 on the TME, we examined its systemic impact on other lymphoid organs during early- and mid-stage tumor development. We identified a higher prevalence of tumor-specific effector CD4 and CD8 T cells in the tumor-draining lymph nodes (tdLNs) of mice bearing CX3CL1 knockout tumors compared to mice bearing parental tumors, as well as increased DCs, particularly cDC1. The cDC1 population expressed antigen-presenting and migratory markers, suggesting these DCs had migrated to the tdLN from tumors. Notably, these systemic changes began early in tumor growth.Conclusions We establish that high CX3CL1 is a feature of cold tumors and inhibits both T cells and DCs, in the tumor itself and systemically. We also demonstrate that the targeted inhibition of CX3CL1 can overcome immunotherapy resistance in cold tumors. This study supports the pursuit of novel therapeutic approaches targeting CX3CL1, particularly in cold tumors where tumor cells produce high levels of CX3CL1.Ethics Approval All animal experiments were approved by the University of Utah Office of Comparative Medicine (IACUC#22-02003)