Increasing evidence indicates that tumors require a constant influx of myelomonocytic cells to support their malignant behavior. This is caused by tumor-derived factors, which recruit and induce functional differentiation of myelomonocytic cells, most of which are macrophages. Although myeloid lineages are the classical precursors of macrophages, B-lymphoid lineages such as B-1 cells, a subset of B-lymphocytes found predominantly in pleural and peritoneal cavities, are also able to migrate to inflammatory sites and differentiate into mononuclear phagocytes exhibiting macrophage-like phenotypes. Here we examined the interplay of B-1 cells and tumor cells, and checked whether this interaction provides signals to influence melanoma cells metastases. Using in vitro coculture experiments we showed that B16, a murine melanoma cell line, and B-1 cells physically interact. Moreover, interaction of B16 with B-1 cells leads to up-regulation of metastasis-related gene expression (MMP-9 and CXCR-4), increasing its metastatic potential, as revealed by experimental metastases assays in vivo. We also provide evidence that B16 cells exhibit markedly up-regulated phosphorylation of the extracellular signal–regulated kinase (ERK) when cocultured with B-1 cells. Inhibition of ERK phosphorylation induced by B-1 cells with inhibitors of MEK1/2 strongly suppressed the induction of MMP-9 and CXCR-4 mRNA expression and impaired the increased metastatic behavior of B16. In addition, constitutive levels of ERK1/2 phosphorylation in B-1 cells are necessary for their commitment to affect the metastatic potential of B16 cells. Our findings show for the first time that B-1 lymphocytes can contribute to tumor cell properties required for invasiveness during metastatic spread. (Cancer Sci 2008; 99: 920–928)