Upon activation, tumor necrosis factor alpha (TNF-α) receptor can engage apoptotic or survival pathways. Inhibition of macromolecular synthesis is known to sensitize cells to TNF-α-induced cell death. It is believed that this sensitization is due to the transcriptional blockade of genes regulated by NF-κB. Nevertheless, such evidence has remained elusive in the nervous system. Here, we show that TNF-α cannot normally induce apoptosis in PC12 cells or cortical neurons. However, cells treated with Actinomycin D (ActD) become susceptible to TNF-α-induced cell death through the activation of caspase-8, generation of tBid and activation of caspase-9 and -3. Analysis of several proteins involved in TNF-α receptor signaling showed no significant downregulation of NF-κB target genes, such as IAPs or FLIP, under such conditions. However, Bcl-xL protein levels, but not those of Bcl-2, Bax and Bak, are reduced by ActD or TNF-α/ ActD treatments. Moreover, Bcl-xL overexpression fully protects cells against TNF-α/ActD-induced cell death. When endogenous levels of Bcl-xL are specifically downregulated by lentiviral-based RNAi, cells no longer require ActD to be sensitive to TNF-α-triggered apoptosis. Furthermore, Bcl-xL downregulation does not affect TNF-α-mediated NF-κB activation. Altogether, our results demonstrate that Bcl-xL, and not Bcl-2, FLIP or IAPs, acts as the endogenous regulator of neuronal resistance/sensitivity to TNF-α-induced apoptosis in an NF-κB-independent manner.