In this chapter, we describe the derivation and characterization of nine hIn this chapter, we describe the derivation and characterization of nine human embryonic stem cells (hESC) (VAL-3 to -11B) from different developmental embryo stages (inner cell mass from a blastocyst, morula, and blastomere from a 3-day embryo) under xeno-free conditions providing the necessary protocols and techniques to carry out their derivation, characterization, and propagation.