The cell-surface straight and branched repeats of N-acetyllactosamine (LacNAc) units, called poly-LacNAc chains, characterize the histo-blood group i and I antigens, respectively. The transition of straight to branched poly-LacNAc chain (i to I) is determined by the I locus, which expresses 3 IGnT transcripts, IGnTA, IGnTB, and IGnTC. Our previous investigation demonstrated that the i-to-I transition in erythroid differentiation is regulated by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). In the present investigation, the K-562 cell line was used as a model to show that the i-to-I transition is determined by the phosphorylation status of the C/EBPalpha Ser-21 residue, with dephosphorylated C/EBPalpha Ser-21 stimulating the transcription of the IGnTC gene, consequently resulting in I branching. Results from studies using adult erythropoietic and granulopoietic progenitor cells agreed with those derived using the K-562 cell model, with lentiviral expression of C/EBPalpha in CD34(+) hematopoietic cells demonstrating that the dephosphorylated form of C/EBPalpha Ser-21 induced the expression of I antigen, granulocytic CD15, and also erythroid CD71 antigens. Taken together, these results demonstrate that the regulation of poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis share a common mechanism, with dephosphorylation of the Ser-21 residue on C/EBPalpha playing the critical role.