In several erythroleukemia cell lines, activation of mitogen-activated protein kinases (MAPK) by phorbol esters or megakaryocyte growth and development factor (MGDF) is required for induction of megakaryocytic phenotype and growth arrest. To support this model, we have examined the effect of a specific inhibitor of this pathway (PD98059) on human CD34⁺ hematopoietic progenitors isolated from cord blood (CB), induced to differentiate along the megakaryocytic lineage in liquid cultures supplemented with rhuMGDF. RhuMGDF induced a sustained activation of MAPK in megakaryocytes and this activation was completely inhibited in the presence of low concentrations of PD98059 (6 to 10 μmol/L). At this concentration, PD98059 induced an increase in cell proliferation, resulting in accumulation of viable cells and a prolongation of the life time of the cultures. This increase correlated with an increase in DNA synthesis rather than with a reduction in apoptosis. This effect was combined with developmental changes indicative of delayed megakaryocytic differentiation: (1) PD98059-treated cells tended to retain markers of immature progenitors as shown by the increased proportion of both CD34⁺ and CD41⁺CD34⁺ cells. (2) PD98059-treated cultures were greatly enriched in immature blasts cells. (3) PD98059 increased megakaryocytic progenitors able to form colonies in semisolid assays. Thus, the MAPK pathway, although not required for megakaryocyte formation, seems to be involved in the transition from proliferation to maturation in megakaryocytes. Inhibition of MAPK activation also led to an increase in the number and size of erythroid colonies without affecting granulocyte/macrophage progenitor numbers suggesting that, in addition to the megakaryocytic lineage, the MAPK pathway could play a role in erythroid lineage differentiation.