Cachexia is a frequent complication of cancer or other chronic diseases. To investigate the pathophysiology of cancer cachexia and pursue treatment options, we developed an in vitro assay of the effects of cancer cell-produced cytokines on primary muscle cells derived from murine skeletal muscle. These studies led to the novel observation that factors secreted by cell lines from prostate cancer and melanoma significantly inhibit differentiation of primary mouse muscle cells. The expression of interleukin (IL) -1, TNF-, and proteolysis-inducing factor (PIF) by cancer cells used in this study suggested their role in preventing myogenic differentiation. Both NF-B binding and transcriptional activity were enhanced in muscle cells treated with conditioned media from cancer cells or with proinflammatory cytokines. Stable expression of IKBSR, a known repressor of NF-B activation, and cellular caspase-8-like inhibitory protein (cFLIP) inhibited activation of NF-B in cancer cell media-treated muscle cells with an accompanying enhancement of myogenic protein expression and differentiation. In contrast, overexpression of antiapoptotic protein Bcl-xL did not protect myoblast cells exposed to the same treatment. Instead, we observed enhanced activation of NF-B in Bcl-xL overexpressing cells. These studies show that the in vitro system recapitulates some of the molecular events causing muscle cachexia and provides the basis for new treatment approaches.--Jiang, Z., Clemens, P. R. Cellular caspase-8-like inhibitory protein (cFLIP) prevents inhibition of muscle cell differentiation induced by cancer cells.