Constitutive phosphorylation of c-kit tyrosine kinase is the major cause of factor-independent proliferation of mast cells. Recently available tyrosine kinase inhibitors have shown marked activity against mast cell lines that carry wild-type c-kit, and some, but not others, carry mutant c-kit. Here we clearly demonstrated that a novel NF-kappaB inhibitor, IMD-0354, restrained factor-independent proliferation of mast cells with c-kit mutations but not of normal mast cells. In HMC-1 cells with the Asp816Val and Val560Gly mutations, we found that NF-kappaB was constitutively activated without exogenous stimulation. When the DNA-binding activity of NF-kappaB was inhibited by treatment with IMD-0354, cell proliferation was completely suppressed. We detected the expression of cyclin D2, D3, and E in HMC-1 cells and observed that cyclin D3 expression was dramatically decreased by treatment with IMD-0354. Abolishing protein kinase C or phosphatidylinositol 3 kinase pathways also inhibited NF-kappaB translocation to the nucleus, indicating the involvement of these signaling cascades in NF-kappaB activation in HMC-1 cells. Our findings indicated that autophosphorylated c-kit receptors induced NF-kappaB activation, resulting in the up-regulation of cyclin D3 expression and cell cycle progression. The observations from the current study suggest a therapeutic potential, in systemic mastocytosis, for compounds that interfere with NF-kappaB signaling.