The addition of burst forming activity (BFA) from mouse spleen conditioned medium (MSCM) to serum-free long-term cultures induced the formation of day 4 BFUE demonstrating that MSCM has an erythropoietin-independent activity on erythroid development. Conditioned medium (CM) from long-term mouse marrow cell cultures was inhibitory to burst formation at concentrations as low as 0.01% when bone marrow cells were used as the external source of BFA, whereas significant levels of inhibition were apparent only at 100-fold greater concentrations of CM in cultures containing MSCM. CM did not inhibit erythropoietin-stimulated erythropoiesis in liquid subcultures of long-term culture cells nor did it stimulate erythroid maturation in the absence of erythropoietin. Adherent cells from the long-term cultures, used as feeder cells, were more active than normal bone marrow cells in enhancing erythroid burst formation.