Objective Brain development is supported by concomitant development of brain vasculature.Prevailing notions of CNS vascularization suggest that blood vessels penetrate passively into brain parenchyma from pial vascular plexuses for meeting metabolic needs of growing neural tissue.It still remains elusive how the brain vasculature forms.Methods In the present study, we characterized the 3-D midbrain vasculature pattern formation during development using in vivo time-lapse confocal imaging of transgenic zebrafish larvae, of which blood endothelial cells express GFP.Results We found that midbrain angiogenic sprouts emanated from the bilateral choroidal vascular plexuses, elongated dorsally, and targeted to the mesencephalic central artery at the midline during 2-5 days post-fertilization (dpf).During later stages, some vessels sprouted from the pial vessels, grew ventrally and anatomosed with the mesencephalic central artery.Furthermore, stereotyped expression patterns of semaphrin 3E and wnt3 were required for this pattern formation of brain vasculature.Further experiments are being performed to examine its detailed molecular mechanisms.