Myocyte enhancer factor 2 (MEF2) transcription factors cooperate with the MyoD family of basic helix-loop-helix (bHLH) transcription factors to drive skeletal muscle development during embryogenesis, but little is known about the potential functions of MEF2 factors in postnatal skeletal muscle. Here we show that skeletal muscle-specific deletion of Mef2c in mice results in disorganized myofibers and perinatal lethality. In contrast, neither Mef2a nor Mef2d is required for normal skeletal muscle development in vivo. Skeletal muscle deficient in Mef2c differentiates and forms normal myofibers during embryogenesis, but myofibers rapidly deteriorate after birth due to disorganized sarcomeres and a loss of integrity of the M line. Microarray analysis of Mef2c null muscles identified several muscle structural genes that depend on MEF2C, including those encoding the M-line-specific proteins myomesin and M protein. We show that MEF2C directly regulates myomesin gene transcription and that loss of Mef2c in skeletal muscle results in improper sarcomere organization. These results reveal a key role for Mef2c in maintenance of sarcomere integrity and postnatal maturation of skeletal muscle.