This monograph will have practical use to those who are interested to establish embryonic stem cells laboratory. Embryonic stem cells are one of the emerging areas in reproductive biotechnologies in buffalo, which could have enormous agricultural and biomedical applications. Besides its immense potential in humans, the stem cell technology has proved to be a boon for farm animals since promising results have been obtained in the fields of gene targeting, cloning, transgenesis, chimera formation and differentiation into germ cells (sperm and oocytes). This book mainly deals with the isolation, culture, characterization and differentiation of embryonic stem cells generated from in vitro fertilized embryos in buffalo and grown on different feeder cells. It will also give the overview of preparation of feeder cells, cryopreservation, in vitro fertilization and parthenogenetic activation.