AbstractTumor cells cultured in three‐dimensional models provide a more realistic and biologically meaningful analysis of the initial phases of cancer development and drug resistance. Several studies have demonstrated that culture of cancer cells in three dimensions induces cellular resistance to a variety of anti‐neoplastic drugs by poorly understood mechanisms. The role of the transcription factor NF‐κB and inhibitors of apoptosis proteins (IAPs) in the onset and development of drug resistance during tumor spheroid growth has not been established. In this work, we found a significant increase in the activity and expression of NF‐κB and its downstream target XIAP (X‐linked IAP) in cancer cells grown as multi‐cellular tumor spheroids. Blocking XIAP expression with RNA interference markedly increased the sensitivity of cancer tumor spheroid cells toward anti‐neoplastic drugs, indicating a role for IAPs in establishing drug resistance. In turn, inhibition of NF‐κB by negative dominants suppressed spheroid formation, whereas overexpression of the upstream kinase IκBKβ increased their growth and resistance. The present data suggested that NF‐κB and its downstream target XIAP were essential for the growth and drug resistance of small avascular tumor. J. Cell. Biochem. 108: 169–180, 2009. © 2009 Wiley‐Liss, Inc.