Structural specification of skeletal muscle is not visually obvious compared to more dramatic examples such as the heart and brain. Though its functional subdivision is not clear to the naked eye, skeletal muscle is a complex and highly organized organ which orchestrates the movement of the body. Skeletal muscles are formed by the assembly of many myofibers with distinct properties, generally classified as slow-twitch and fast-twitch fibers. The physiological distinctions between specific muscle fiber types are determined by expression of fiber type-specific contractile protein isoforms (e.g. myosin heavy chain) and metabolic enzyme profiles that support the mechanistic requirement. Each mammalian skeletal muscle is a heterogeneous bundle of different types of myofibers, allowing the same muscle to respond to a wide spectrum of physiological activities. A working unit that controls the movement of skeletal muscle is the motor unit. The motor unit consists of a motor neuron and myofibers of similar functional properties, i.e. a slow type motor neuron innervates slow and more oxidative myofibers whereas a fast type motor neuron innervates fast and more glycolytic myofibers. This functional coupling of the motor unit is established during late gestation to early postnatal stages in mammals. Recent studies utilizing genetically engineered animal models have revealed many regulatory factors involved in muscle fiber type specification and the hierarchical organization of motor neurons. The molecular mechanisms governing the functional matching of motor neurons and skeletal myofibers, however, still remain poorly understood. This short review summarizes the representative studies which revealed the regulatory networks responsible for achieving the organized development of skeletal muscles and motor neurons and discusses the prospect of identifying the mechanisms establishing the motor unit during mammalian development.