Accumulating evidence suggests that activin A/erythroid differentiation factor (EDF), a homodimer of beta A chain, is a physiologic hematopoietic factor, particularly of erythroid lineage. Media conditioned by phorbol myristate acetate (PMA)-stimulated murine bone marrow stromal cell lines, MC3T3-G2/PA6 and ST2, contained activin A/EDF, assayed as the activity inducing erythroid differentiation of an activin A/EDF-responsive murine erythroleukemia cell clone F5. Follistatin, a protein specifically binding to activin A/EDF, abolished this erythroid differentiation. Northern blot analysis showed that PMA rapidly increased beta A chain messenger RNA levels in MC3T3-G2/PA6 and ST2 cells. In a search for natural stimulators, we found that tumor necrosis factor-alpha, in itself and synergistically with interleukin-1 beta, induced activin A/EDF production in both cell lines. These results indicate that stromal cells produce activin A/EDF in bone marrow.