The function of theDrosophila mef2gene, a member of the MADS box supergene family of transcription factors, is critical for terminal differentiation of the three major muscle cell types, namely somatic, visceral, and cardiac. During embryogenesis,mef2undergoes multiple phases of expression, which are characterized by initial broad mesodermal expression, followed by restricted expression in the dorsal mesoderm, specific expression in muscle progenitors, and sustained expression in the differentiated musculatures. In this study, evidence is presented that temporally and spatially specificmef2expression is controlled by a complex array ofcis-acting regulatory modules that are responsive to different genetic signals. Functional testing of ∼12 kb of 5′ flanking region of themef2gene showed that the initial widespread mesodermal expression is achieved through a 280-bptwist-dependent enhancer. The subsequent dorsal mesoderm-restrictedmef2expression is mediated through a 460-bpdpp-responsive regulatory module, which involves the function of the Smad4 homologMedeaand contains several binding sites for Medea and Mad. The analysis also showed that regulatedmef2expression in the caudal and trunk visceral mesoderm, which give rise to longitudinal and circular gut musculatures, respectively, is under the control of distinct enhancer elements. In addition,mef2expression in the cardioblasts of the heart is dependent upon at least two distinct enhancers, which are active at different periods during embryogenesis. Moreover, multiple regulatory elements are differentially activated for specific expression in presumptive muscle founders, prefusion myoblasts, and differentiated muscle fibers. Taken together, the presented data suggest that specific expression of themef2gene in myogenic lineages in theDrosophilaembryo is the result of multiple genetic inputs that act in an additive manner upon distinct enhancers in the 5′ flanking region.