Objective: Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for selfrenewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently, stem cell culture has been carried out using feeder cells, and culture media, that contain animal products. Recent advances in technology have opened up the possibility of both xenofree and feeder-free culture of stem cells, essential conditions for the use of stem cells for clinical purposes. Materials and Methods: The derivation of hES cells at King’ s College London (London, UK) is under license from the UK Human Fertilisation and Embryology Authority (HFEA; research license number R0133) and also has local ethical approval (UK National Health Service Research Ethics Committee Reference 06/Q0702/90). No financial inducements are offered for donation. In accordance with HFEA regulations, a sample of each line that is derived is deposited in the UK Stem Cell Bank for distribution to academic and research centers internationally. Fresh embryos for the derivation were obtained from the Guy’ s Assisted Conception Unit (ACU; London, UK) pre-implantation genetic diagnostics (PGD) program. Cryopreserved embryos no longer wanted for therapeutic use by patients were from both Guy’s ACU as well as external units, obtained through the human embryonic stem cell co-ordinator’s (hESCCO) network. Results: Detailed protocols are developed for deriving human embryonic stem cell lines in xeno-free conditions from cryopreserved embryos. From derivation to cryopreservation of fully characterized initial stocks takes 3-4 months. These protocols served as the basis for standard operating procedures (SOPs), with both operational and technical components, that we set to meet good manufacturing practice (GMP) and UK regulatory body requirements for derivation of clinical-grade cells. Conclusion: As such, these SOPs were used in our current GMP-compliant facility to derive hESEs cell lines ab initio, in an animal product-free environment; these lines are suitable for research and potentially for clinical use in cell therapy. So far, we have derived eight clinical-grade lines, which will be freely available to the scientific community after submission/accession to the UK Stem Cell Bank.