Notch signaling is mediated by cell-cell interactions and is critical for cell fate determination in many species. Recently, a family of mastermind-like (MAML) transcriptional co-activator genes was identified that encode proteins that cooperate with Notch and CSL to activate transcription. Here, we review our current understanding of the roles of the MAML proteins in Notch signaling, and their involvement in certain human cancers. The mounting biochemical and functional evidence indicate that the MAML genes are critical components of the Notch signaling pathway, likely regulating cellular events involved in both normal development and oncogenesis.