hESCs (human embryonic stem cells) are pluripotent cells derived from the ICM (inner cell mass) of blastocysts that can be used to derive several kinds of cells of the human body for the treatment of some previously untreated diseases. In considering the future use of hESCs in regenerative medicine and cell-therapy programmes, several research centres have begun projects involving the derivation of hESC lines using spare human embryos from IVF (in vitro fertilization) cycles. In some stem-cell banks, such as ours, the law also permits us to obtain these cell lines. The low availability of spare IVF human embryos, and the low rate of success in the derivation of hESC lines, give these embryos a great research value that limits experiments with new techniques. The use of murine embryos would be a good model with which to do research to discover the best methodologies to use in order to derive new hESC lines. The aim of the present study was to evaluate a new method of isolation of the ICM and derivation of ESC lines in a murine blastocyst model using laser drilling to eliminate the trophectoderm cells and compare it with the usual control method consisting of culturing the whole murine blastocyst. We also tested the adhesion and growth of primary colonies of mESCs (murine ESCs) over two different growth surfaces, namely an MEF (inactive murine fibroblastic feeder layer) or gelatin-coated dishes, in order to achieve the best culture conditions for future derivation of human stem-cell lines for application in human transplantation.