Thrombopoietin (TPO) is a lineage-dominant hematopoietic cytokine that regulates megakaryopoiesis and platelet production. The major site of TPO biosynthesis is the liver. Despite easily detectable levels of liver TPO mRNA, the circulating TPO serum levels are very low. We have observed that translation of TPO mRNA is inhibited by the presence of inhibitory elements in the 5'-untranslated region (5'-UTR). Alternative promoter usage and differential splicing generate at least three TPO mRNA isoforms that differ in the composition of their 5'-UTR. Using mutational analysis we show that physiologically the translation of these TPO mRNA isoforms is strongly inhibited by the presence of AUG codons, which define several short open reading frames (ORFs) in the 5'-UTR and suppress efficient initiation at the physiologic start site. The two regularly spliced isoforms, which account for 98% of TPO mRNA, were almost completely inhibited, whereas a rare splice variant that lacks exon 2 can be more efficiently translated. Thus, inhibition of translation of the TPO mRNA is an efficient mechanism to prevent overproduction of this highly potent cytokine.