Studies in zebrafish and rats show that embryonic ethanol exposure at low-moderate concentrations stimulates hypothalamic neurons expressing hypocretin/orexin (Hcrt) that promote alcohol consumption, effects possibly involving the chemokine Cxcl12 and its receptor Cxcr4. Our recent studies in zebrafish of Hcrt neurons in the anterior hypothalamus (AH) demonstrate that ethanol exposure has anatomically specific effects on Hcrt subpopulations, increasing their number in the anterior AH (aAH) but not posterior AH (pAH), and causes the most anterior aAH neurons to become ectopically expressed further anterior in the preoptic area (POA). Using tools of genetic overexpression and knockdown, our goal here was to determine whether Cxcl12a has an important function in mediating the specific effects of ethanol on these Hcrt subpopulations and their projections. The results demonstrate that the overexpression of Cxcl12a has stimulatory effects similar to ethanol on the number of aAH and ectopic POA Hcrt neurons and the long anterior projections from ectopic POA neurons and posterior projections from pAH neurons. They also demonstrate that knockdown of Cxcl12a blocks these effects of ethanol on the Hcrt subpopulations and projections, providing evidence supporting a direct role of this specific chemokine in mediating ethanol’s stimulatory effects on embryonic development of the Hcrt system.