Children with Down syndrome (DS) are at increased risk for acute myeloid leukemias (ML-DS) characterized by mixed megakaryocytic and erythroid phenotype and by acquired mutations in the GATA1 gene resulting in a short GATA1s isoform. The chromosome 21 microRNA (miR)-125b cluster has been previously shown to cooperate with GATA1s in transformation of fetal hematopoietic progenitors. In this study, we report that the expression of miR-486-5p is increased in ML-DS compared with non-DS acute megakaryocytic leukemias (AMKLs). miR-486-5p is regulated by GATA1 and GATA1s that bind to the promoter of its host gene ANK1. miR-486-5p is highly expressed in mouse erythroid precursors and knockdown (KD) in ML-DS cells reduced their erythroid phenotype. Ectopic expression and KD of miR-486-5p in primary fetal liver hematopoietic progenitors demonstrated that miR-486-5p cooperates with Gata1s to enhance their self renewal. Consistent with its activation of AKT, overexpression and KD experiments showed its importance for growth and survival of human leukemic cells. Thus, miR-486-5p cooperates with GATA1s in supporting the growth and survival, and the aberrant erythroid phenotype of the megakaryocytic leukemias of DS.