The transcription factor C/EBPα is essential for myeloid differentiation and is frequently dysregulated in acute myeloid leukemia. Although studied extensively, the precise regulation of its gene by upstream factors has remained largely elusive. Here, we investigated its transcriptional activation during myeloid differentiation. We identified an evolutionarily conserved octameric sequence, CCCAGCAG, ~100 bases upstream of the CEBPA transcription start site, and demonstrated through mutational analysis that this sequence is crucial for C/EBPα expression. This sequence is present in the genes encoding C/EBPα in humans, rodents, chickens, and frogs and is also present in the promoters of other C/EBP family members. We identified that ZNF143, the human homolog of the Xenopus transcriptional activator STAF, specifically binds to this 8-bp sequence to activate C/EBPα expression in myeloid cells through a mechanismthat is distinctfromthat observed in liver cellsandadipocytes. Altogether, our data suggest that ZNF143 plays an important role in the expression of C/EBPα in myeloid cells.