Notch signaling is critical for skeletal muscle development and regeneration, permitting the expansion of progenitor cells by preventing premature differentiation. We have interrogated the pathways through which ligand‐mediated signaling inhibits myogenesis by identifying Notch target genes and assessing their impact on differentiation in vitro. Notch activation led to the robust induction of the transcriptional repressors Hey1 and HeyL in myoblasts, but only constitutive expression of Hey1 blocked myogenesis. siRNA‐mediated knockdown of Hey1 had no effect on Notch's ability to inhibit differentiation, suggesting the existence of additional, possibly redundant pathways. We identified 82 genes whose expression was activated when C2C12 myoblasts were cultured in the presence of the Notch ligand Dll4. One of these, MyoR, is a novel Notch‐responsive gene, whose protein product is known to repress myogenesis in vitro. siRNA‐mediated knockdown of MyoR alone, or in combination with Hey1, was also ineffective at rescuing differentiation in the presence of Dll4. Our data support a model in which Notch signaling inhibits myogenesis through multiple pathways, two of which are defined by the Notch target genes Hey1 and MyoR. J. Cell. Physiol. 218: 84–93, 2009. © 2008 Wiley‐Liss, Inc.