We recently identified two siblings afflicted with idiopathic, autosomal recessive aplastic anemia. Whole-exome sequencing identified a novel homozygous missense mutation in thrombopoietin (THPO, c.112C>T) in both affected siblings. This mutation encodes an arginine to cysteine substitution at residue 38 (R38C), or residue 17 (R17C) excluding the 21 amino acid signal peptide, of THPO receptor binding domain (RBD). THPO has four conserved cysteines in its RBD that form two disulfide bonds. Our in silico modeling predicts that introduction of a fifth cysteine may disrupt normal disulfide-bonding to cause poor receptor-binding. In functional assays, the mutant-THPO-containing media shows 2-3 fold reduced ability to sustain UT7-TPO cells, which require THPO for proliferation. Both parents and a sibling with heterozygous R17C change have reduced platelet counts, while a sibling with wildtype sequence has normal platelet count. Thus the R17C partial loss-of-function allele results in aplastic anemia in the homozygous state, and mild thrombocytopenia in the heterozygous state in our family. Together with the recent identification of THPO receptor (MPL) mutations and the effects of THPO agonists in aplastic anemia, our results have clinical implications in the diagnosis and treatment of patients with aplastic anemia and highlight a role for THPO-MPL pathway in hematopoiesis in vivo.