Deciphering the cellular signals leading to cardiac muscle assembly is a major challenge in ex vivo tissue regeneration. For the first time, we demonstrate that pulsatile interstitial fluid flow in three-dimensional neonatal cardiac cell constructs can activate ERK1/2 sixfold, as compared to static-cultivated constructs. Activation of ERK1/2 was attained under physiological shear stress conditions, without activating the p38 cell death signal above its basic level. Activation of the ERK1/2 signaling cascade induced synthesis of high levels of contractile and cell-cell contact proteins by the cardiomyocytes, while its inhibition diminished the inducing effects of pulsatile flow. The pulsed medium-induced cardiac cell constructs showed improved cellularity and viability, while the regenerated cardiac tissue demonstrated some ultra-structural features of the adult myocardium. The cardiomyocytes were elongated and aligned into myofibers with defined Z-lines and multiple high-ordered sarcomeres. Numerous intercalated disks were positioned between adjacent cardiomyocytes, and deposits of collagen fibers surrounded the myofibrils. The regenerated cardiac tissue exhibited high density of connexin 43, a major protein involved in electrical cellular connections. Our research thus demonstrates that by judiciously applying fluid shear stress, cell signaling cascades can be augmented with subsequent profound effects on cardiac tissue regeneration.