Objective. To determine novel genes regulated by tumor necrosis factor α (TNFα) signaling in primary rheumatoid arthritis synovial fibroblasts (RASFs). Methods. Oligonucleotide microarrays were used to measure gene expression levels in 6 independent replicate samples of RASFs. RASFs were transfected for 18 hours with AdIκB-dominant negative (AdIκB-DN) (n = 3) or with control AdTet expressing the reverse tetracycline trans-activator (n = 3). The cells were stimulated for 3 hours with TNFα, and total RNA was prepared. Several novel parametric and nonparametric methods were used to rank genes in terms of the magnitude and significance of intergroup differences. Microarray expression differences were confirmed by real-time quantitative reverse transcription-polymerase chain reaction. Small interfering RNA (siRNA) was used to specifically down-modulate microarray-identified genes to demonstrate their role in the promotion of apoptosis, proliferation, or matrix metalloproteinase (MMP) expression. Results. Blocking of NF-κB by AdIκB-DN was associated with a down-modulation of antiapoptosis genes, including BIRC-3, and several novel genes, including GG2-1, a TNFα-inducible FLIP-like gene. Other families of genes that were significantly down-regulated by AdIκB-DN included cytokines/chemokines (interleukin-1β [IL-1β], IL-8, IL-15, and RANTES), adhesion molecule (vascular cell adhesion molecule 1, intercellular adhesion molecule 1), and unique genes that have not previously been reported to be regulated by TNFα in RA. Inhibition of the GG2-1 gene using the siRNA technique resulted in significantly enhanced apoptosis, decreased proliferation, and decreased production of MMP-1 in TNFα-stimulated RASFs. Conclusion. These studies provide a comprehensive analysis of genes that are differentially regulated by TNFα signaling and NF-κB nuclear translocation in RASFs and demonstrate methods for confirming the expression and functional significance of such genes.