Sexual dimorphism of white matter has not been considered important, the assumption being that sex hormones are not essential for glial development. We recently showed exogenous hormones in vivo differentially regulate in male and female rodents the life span of oligodendrocytes (Olgs) and amount of myelin (Cerghet et al. [2006] J. Neurosci. 26:1439-1447). To determine which hormones regulate male and female Olg development, we prepared enriched Olg cultures grown in serum-free medium with estrogen (E2), progesterone (P2), and dihydrotestosterone (DHT) or their combinations. P2 significantly increased the number of Olgs in both sexes, but more so in females; E2 had minor effects on Olg numbers; and DHT reduced Olgs numbers in both sexes, but more so in females. Combinations of hormones affected Olg numbers differently from single hormones. The change in Olg numbers was due to changes not in proliferation but rather in survival. P2 increased pAKT by many-fold, but MAPK levels were unchanged, indicating that activation of the Akt pathway by P2 is sufficient to regulate Olg differentiation. DHT reduced pAkt in both sexes but differentially increased pMAPK in males and decreased it in females. Stressing Olgs reveals that both sexes are protected by P2, but females are slightly better protected than males. Females always showed greater differences than males regarding changes in Olg numbers and in signaling molecules. Given the greater fluctuation of neurosteroids in women than in men and the higher incidence of multiple sclerosis (MS) in women, these sexually dimorphic differences may contribute to differences in male and female MS lesions.