Abstract We previously reported that breast cancer (BC) generates metastasis-promoting regulatory B cells (CD25+ tumor-evoked Bregs). Because phenotypically these cells resembled bone marrow (BM) Rag+ B-cell precursors, such as large Pre-B cells, we hypothesized whether cancer uses them to generate Bregs. To investigate this idea, we used mouse and human BM aspirates, human PBMCs from BC patients, and mice with different orthotopic cancers, such as highly metastatic 4T1 breast cancer cells and EMT6. We report that breast cancer indeed uses BM B-cell precursors to generate Bregs. However, to do this, cancer expresses thymic stromal lymphopoietin (TSLP). Mechanistically, cancer-produced TSLP downregulates surface expression of CXCR4 and α4β1 (VLA-4) of B-cell precursors, thereby impairing their BM retention and increasing their exit into the circulation. TSLP also enables survival and proliferation of these BM emigrants, while other factors such as 5-lipoxygenase metabolites convert them into Bregs. The loss of TSLP expression in cancer cells alone or TSLPR deficiency in B cells not only reduce pre-B cells in the circulation, but also retard lung metastasis. Overall, our data reveal a previously unknown function of cancer-produced TSLP as inducers of premature B-cell precursor emigration from BM to support Breg generation. The results also suggest that the TSLP-pre-B cell axis can be an attractive therapeutic target. Citation Format: Emeline Ragonnaud, Kanako Moritoh, Monica Bodogai, Soizic Garaud, Chen Chen, Xin Wang, Karen Willard-Gallo, Arya Biragyn. Cancer targets early B-cell precursors to generate metastasis-promoting Bregs by promoting their premature emigration from the bone marrow and expansion in the circulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4499.