Mircoglia are specialized mononuclear phagocytes within the central nervous system (CNS). Their embryonic origins and routes of entry to the CNS remain controversial. In this study, we used Iba1 as a specific marker for microglial progenitors in mouse embryos and employed multiple immunohistochemical localization and tissue transplantation to establish (1) their routes of entry to the CNS and (2) their origin. Iba1+ microglial progenitors were found in the mesenchyme at E9.5, and started to appear in the neural tube at E10.5. By E13.5, significant numbers of Iba1+ cells were observed in the mantle layer. Examination of sectioned tissues suggested that Iba1+ cells entered the neural tube through its basal surface, apical surface or blood vessels. When fragments of E11.5 liver tissues genetically labeled with eGFP were transplanted to the mesenchyme next to the unlabeled E10.5 neural tube, eGFP+Iba1+ cells were found within the neural tube one day after transplantation, implicating that Iba1+ cells from the embryonic liver were able to migrate through the mesenchyme to enter the neural tube. In conclusion, microglial progenitors expressing Iba1 enter the neural tube through multiple sites, and the embryonic liver could represent a source of origin.The work was supported by the General Research Fund from the Research Grants Committee of the Hong Kong Special Administrative Region, China (Project No. 461909).