Human embryonic stem cells (hESCs) are self-renewing, pluripotent cells derived from the inner cell mass of blastocysts, early-stage embryos, or blastomeres. hESCs can be propagated indefinitely in an undifferentiated state in vitro and have the ability to differentiate into all cell types of the body. Therefore, these cells can potentially provide an unlimited source of cells and hold promise for transplantation therapy, regenerative medicine, drug screening and discovery, and basic scientific research. Surplus human embryos donated for hESC derivation are extremely valuable, and inefficient derivation of hESCs would be a terrible waste of human embryos. Here, we describe a method for isolating hESC lines from human blastocysts with high efficiency. We also describe the methods for excising differentiated areas from partially differentiated hESC colonies and re-isolating undifferentiated hESCs from extremely differentiated hESC colonies.