Binding of tumor necrosis factor‐α (TNF‐α) to its receptor activates IKK complex, which leads to inducement of NF‐κB activity. Here we report that activation of Mpl ligand is also linked to IKK and NF‐κB activity. Mpl ligand, also known as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF), induces megakaryocyte differentiation and inhibition of mitotic proliferation, followed by induction of polyploidization and fragmentation into platelets. The latter process is often observed in megakaryocytes undergoing apoptosis. Treatment of a Mpl ligand‐responding megakaryocytic cell line with this cytokine led to an immediate, transient increase in IKK activity followed by a profound decrease in this kinase activity over time. This decrease was not due to an effect on the levels of the IKK regulatory components IKKα and IKKβ. Proliferating megakaryocytes displayed a constitutive DNA‐binding activity of NF‐κB p50 homodimers and of NF‐κB p50–p65 heterodimers. As expected, reduced IKK activity in Mpl ligand‐treated cells was associated with a significant reduction in NF‐κB DNA binding activity and in the activity of a NF‐κB‐dependent promoter. Our study is thus the first to identify a constitutive NF‐κB activity in proliferating megakaryocytes as well as to describe a link between Mpl receptor signaling and IKK and NF‐κB activities. Since a variety of proliferation‐promoting genes and anti‐apoptotic mechanisms are activated by NF‐κB, retaining its low levels would be one potential mechanism by which inhibition of mitotic proliferation is maintained and apoptosis is promoted during late megakaryopoiesis. J. Cell. Biochem. 85: 523–535, 2002. © 2002 Wiley‐Liss, Inc.