In the anterior pituitary, S100β protein (S100β) has been assumed to be a marker of folliculo-stellate cells, which are one of the non-hormone-producing cells existing in the parenchyma of the adult anterior lobe and are composed of subpopulations with various functions. However, recent accumulating studies on S100β-positive cells, including non-folliculo-stellate cells lining the marginal cell layer (MCL), have shown the novel aspect that most S100β-positive cells in the MCL and parenchyma of the adult anterior lobe are positive for sex determining region Y-box 2 (SOX2), a marker of pituitary stem/progenitor cells. From the viewpoint of SOX2-positive cells, the majority of these cells in the MCL and in the parenchyma are positive for S100β, suggesting that S100β plays a role in the large population of stem/progenitor cells in the anterior lobe of the adult pituitary. Reportedly, S100β/SOX2-double positive cells are able to differentiate into hormone-producing cells and various types of non-hormone-producing cells. Intriguingly, it has been demonstrated that extra-pituitary lineage cells invade the pituitary gland during prenatal pituitary organogenesis. Among them, two S100β-positive populations have been identified: one is SOX2-positive population which invades at the late embryonic period through the pituitary stalk and another is a SOX2-negative population that invades at the middle embryonic period through Atwell’s recess. These two populations are likely the substantive origin of S100β-positive cells in the postnatal anterior pituitary, while S100β-positive cells emerging from oral ectoderm-derived cells remain unclear.