MicroRNA (miR) regulates hematopoiesis through targeting different genes post-transcriptionally. We have recently shown that Tip110 expression is downregulated during hematopoietic stem cell differentiation. However, the underlying mechanisms are not known. In this study, we identified a conserved miR-124-binding site on the Tip110 3′-untranslated region (3′-UTR) and showed that Tip110 was downregulated by miR-124 through its 3′-UTR. We then examined the relationship among miR-124 and Tip110 expression and differentiation of human cord blood CD34+ cells. We found that miR-124 was expressed in a low level in human cord blood CD34+ cells, but it was considerably upregulated during culturing and differentiation of these cells. Moreover, we demonstrated that miR-124 expression decreased Tip110 expression and promoted differentiation of human cord blood CD34+ cells, while miR-124 knockdown increased Tip110 expression, slowed down differentiation of human cord blood CD34+ cells, and caused an expansion of hematopoietic progenitor cells in vitro. Finally, we used mouse embryonic fibroblasts derived from Tip110 transgenic mice, performed the exon array analysis, and found that Tip110 altered a number of genes in the hematopoiesis pathways. Dnmt3a as de novo methyltransferase was also significantly upregulated. That miR-124 was markedly upregulated during human cord blood CD34+ cell differentiation could be the result of direct loss of its promoter methylation from Dnmt3a. Taken together, our study demonstrates that miR-124 regulates Tip110 expression and differentiation of human cord blood CD34+ cells and suggests important roles of miR-124/Tip110 in hematopoiesis.