In contrast to mature spleen cells and macrophages, which produce colony-stimulating factor (CSF) in response to lipopolysaccharide (LPS), murine bone marrow (BM) cells do not respond to LPS alone. However, when BM cells are treated with LPS and the tumor-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) simultaneously, they generate significant levels of CSF. The non-tumor-promoter 4-O-methyl-TPA will not replace TPA, and if BM cells from C3H/HeJ mice are employed, no CSF is produced after stimulation with LPS and TPA. Lipid A is as effective as LPS in stimulating BM cells in the presence of TPA, but other mitogens, such as phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM), are ineffective. B-lymphocytes may be the main source of the CSF from BM cells, since BM cells adherent to surfaces coated with goat antimouse immunoglobulin produced CSF in amounts similar to those produced by unseparated BM cells after stimulation with LPS and TPA. Finally, the CSFs produced by BM cells under these experimental conditions were identified as belonging to the GM-CSF and G-CSF subclasses. We interpret these results as suggesting that B cells present in the BM, as opposed to mature spleen cells and macrophages, require at least two signals for CSF production.