During development, the interaction between tenocytes and myotubes leads to the formation of highly specialized muscle-tendon structural interfaces: myotendinous junctions (MTJs). Structural integrity of MTJs is critical for force transmission from contracting muscle through tendon to bone. We recently developed an in vitro model of three-dimensional (3-D) skeletal muscle-tendon constructs to address mechanisms of the MTJs development. We hypothesized that engineered in vitro 3-D skeletal muscle-tendon constructs would develop MTJs ultrastructurally resembling those found during fetal development in vivo. To test this hypothesis we compared MTJs structures in vivo to those developed in 3-D skeletal muscle constructs co-cultured with engineered self-organized tendon constructs (SOT), or segments of adult (ART) or fetal rat tail (FRT) by means of electron microscopy. Our study showed that at sites of termination some of the myofibers of the engineered 3-D skeletal muscle-FRT and -SOT constructs displayed emerging finger-like sarcolemmal projections surrounded by collagen fibers. These structures resemble fetal MTJs in vivo. Muscle-ART constructs did not develop MTJs. Muscle-FRT constructs in addition to muscle and tendon also demonstrated well developed cartilage, possessing high potential for development into bone. The muscle-FRT construct model could be used for studies of developmental mechanisms involved in the establishment of interfaces among all four muscular-skeletal tissues: muscle, tendon and cartilage/bone.