During spinal cord development, endogenous progenitors expressing nestin can migrate into the target and differentiate into neurons and other glial cells. Microglial cells can also be derived from nestin+ progenitor cells, even in the adult brain. Knockdown of Jak kinase 3 (Jak3) signaling can increase neurogenesis with longer neurite outgrowth in cortical progenitor cells. This study investigated the effect of Jak3 signaling on differentiation from nestin+ progenitor cells using E13.5 spinal progenitor cell cultures. In growth factors-enriched culture, developing neurons could not survive after several days and also a significant proportion of nestin-expressing cells transformed into ameboid Iba1+ microglial cells, which increased exponentially after 5 days. This microgliogenesis was predominantly regulated by Jak3 signaling, which was confirmed by transcription factors responsible for microgliogenesis, and microglial migration and phagocytosis, such as Pu.1, Irf8, and Runx1. Jak3 inhibition also significantly increased the Tuj1+ growing neurites with little microglial activation. These results indicated that neuronal and microglial cell differentiation was regulated primarily by Jak3 signaling and the developing neurons and neurite outgrowth might also be regulated by Jak3-dependent microglial activity.