Clones of myeloid leukemic cells varying in their competence for induction of differentiation have been continuously grown in serum-free medium. In the medium used, which contained transferrin, the growth rates of these cells were nearly similar to those found in serum- containing medium. The clones also maintained in this medium their competence for induction of differentiation by the normal macrophage and granulocyte differentiation-induction protein MGI-2, the steroid dexamethasone, and lipopolysaccharide. In contrast to the results with these inducters, some clones continuously cultured in a serum-free medium showed a gain of inducibility by insulin and another clone a gain of inducibility by the tumor promoter 12-O-tetradecanoylphorbol-13- acetate in low serum and serum-free medium. Induction of differentiation by these two compounds was therefore inhibited in these clones by the presence of serum. It is suggested that serum-free medium may also show the existence of other inducers of differentiation not detected in serum-containing medium and that these results are relevant to the possible therapeutic use of compounds such as insulin for the induction of normal differentiation in leukemic cells in vivo.