Congenital and acquired surgical conditions represent a major cause of morbidity and mortality during the first years of life and childhood. In those complex conditions, prosthetic materials are used because of the lack of biocompatible tissues able to replace or regenerate damaged organs. Besides the risk of infection, the major drawback of using a prosthetic patch closure is the risk of dislodgment and subsequent recurrence of the initial problem. Moreover, foreign body reactions and implant rejection occur when synthetic polymers are used. Regeneration of natural tissue from living cells to restore damaged tissues and organs is the main purpose of regenerative medicine. This relatively new field has emerged by the combination of tissue engineering and cell transplantation as a possible strategy for the replacement of damaged organs or tissues. So far, most of the attention has been focused on degenerative diseases such as Parkinson’s or Alzheimer’s, while very little has been done for the treatment of congenital conditions. However, the knowledge acquired in the last years from stem cell (SC) biology and regenerative medicine strategies could lead to new ways of repairing or replacing injured organs and systems, even during fetal development, and therefore, pediatric patients could largely benefit from the evolution of this new exciting field. In order to give rise to a new functional organ-like structure, several variables, such as local environment, nutrients, and metabolites, are pivotal. These variables, in the context of tissue engineering, are mainly dependent on the provision of a three-dimensional growth structure termed “scaffold.”1 Scaffolds can be either natural of synthetic. Natural scaffolds are essentially bioactive but lack mechanical strength. Synthetic scaffolds lack bioactivity, are mechanically strong and can be engineered with bespoke macrostructure and microstructure, which has the potential to enhance cellular growth and organogenesis.2 Scaffolds could ultimately represent the exclusive tool required for tissue engineering, and several attempts to generate whole organs such as liver have been conducted using structures with vascular channels to ensure an adequate network of vascular supply.3 Major developments in regenerative medicine have been achieved following the discovery of cells which can be isolated and expanded in number outside the body. SCs are unspecialized cells with the capacity to both self-renew, and give rise to multiple mature specialized cell types through asymmetric cell division.4 There are three main sources of SCs in humans and animals: embryonic, fetal, and adult tissue. Adult SCs have a limited cellular regeneration, or turnover, this could represent a limitation for tissue engineering applications, where a large number of cells are necessary.5 SCs they can be identified in many adult mammalian tissues, such as bone marrow (BM), skeletal muscle, skin, and adipose tissue, where they contribute to the replenishment of cells lost through normal cellular senescence or injury.6 10 In contrast, SCs derived from embryonic sources have the ability to give rise to cells that not only proliferate and replace themselves indefinitely, but also have the potential to form any cell type.11 , 12 Embryonic stem (ES) cells are derived from the inner cell mass of pre-implantation embryo, they are pluripotent, and demonstrate germ-line transmission in experimentally produced chimeras.13 , 14 More recently, cells with intermediate potency have derived from the amniotic fluid (fetal SCs)15 and from adult SCs which have been reprogrammed using various factors implicated in the maintenance of pluripotency in ES cells.16 This chapter would like to offer an insight on the latest evolution of SCs, with a glance at their possible application for regenerative medicine, and recent clinical translations, particularly in the pediatric surgery field.