The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is crucial for immune responses and skeletal development. Work in recent years has shown that various members of the NF-κB family are viable targets to regulate activity and survival of bone cells and hence bone metabolism. In this regard, deletion of upstream kinases or distal NF-κB subunits resulted with bone deformities. Thus, it has become increasingly apparent that detailed investigation of NF-κB in bone cells may provide opportunities to design new therapeutic modalities. In this chapter we present modified methodology describing efficient approaches to regulate the NF-κB pathway in vitro and in vivo to assess its function in bone cells and tissues.