The present review will discuss new insights of myogenesis that begins when embryonic mononucleated progenitor cells become committed to myogenic lineage and subsequently proliferate and fuse to form multinucleated contracting skeletal muscle fibers, responsible for generation of force and movement. This review will focus mostly on the influence of specific myogenic transcription factors on skeletal muscle differentiation and on the compartmentalized expression of nicotinic acetylcholine receptors and acetylcholinesterase at specific muscle fiber microdomains. Experiments using cultured muscle model obtained from embryonic or adult animals, have provided detailed information on myogenesis. Differentiated cultured skeletal muscle fibers contract spontaneously and preserve several properties of in vivo multinucleated muscle fiber, including the expression of specific myogenic transcription factors as well as the compartmentalized synthesis and expression of neuromuscular synaptic proteins around individual nuclei. Besides, cultured muscle cells express multiple receptors coupled to G protein, including muscarinic acetylcholine receptors. Considering that many aspects of the present knowledge about the development and differentiation of muscle fiber and formation of the neuromuscular synapse were established in studies using muscles cultures, protocols of primary tissue-cultured skeletal muscle obtained either from embryonic myoblasts or adult satellite cells will be presented.