Recent studies have revealed the essential role of the receptor activator of nuclear factor κB (NF‐κB) ligand (RANKL) in osteoclast differentiation and activation. Adenovirus vector could efficiently transduce genes into RAW264.7 cells, which differentiate into osteoclast‐like multinucleated cells in the presence of RANKL. The role of NF‐κB and c‐jun N‐terminal kinase (JNK) activation in RANKL‐induced osteoclast differentiation was investigated using an adenovirus vector carrying the dominant negative IκB kinase 2 gene (AxIKK2^DN) or dominant negative MKK7 gene (AxMKK7^DN). IKK2^DN and MKK7^DN overexpression in RAW cells specifically suppressed the NF‐κB activation and JNK activation in response to RANKL, respectively, without affecting other signaling pathways. Either inhibition of NF‐κB or JNK pathways dose‐dependently inhibited osteoclast formation induced by RANKL. These results suggest that both NF‐κB and JNK activation are independently required for osteoclast differentiation.