Human embryonic stem cell lines are usually derived from human embryos that have become excess to clinical needs in assisted reproduction programs, whether because the couple in question has completed their family or because the embryo was found to be clinically unsuitable for transfer due to severe genetic condition (in case of pre-implantation genetic diagnosis, PGD). Culturing embryos to a blastocyst stage (5-6 days after IVF) before embryo transfer or cryopreservation instead of earlier commonly used 8-cell stage (3 days after IVF) calls for new methods for embryo cryopreservation and allows higher efficiencies for the actual stem cell derivation. Despite the vast advances in other fields of embryonic stem cell research, methods for derivation of new lines have not changed much over the years, mainly due to scarcity of embryos limiting experimentation. We describe here methods required to derive new embryonic stem cell lines starting from the initial cryopreservation of an embryo and finishing with a new cell line. We cover embryo cryopreservation and warming using a highly efficient vitrification method, the production of feeder cells and feeder plates, as well as embryo handling, plating and critical early passages, including earliest possible cryopreservation of putative stem cells using vitrification.