Leukemia (2018) 837–854 diseases, suggesting that mutations identified in hematopoietic stem cells (HSCs) contribute to disease development. 1–5 However, the mechanisms by which age-induced stem cell mutations cause clonal expansion of HSCs are largely unknown. Recently, acquired somatic mutations in TP53 gene were identified in the blood of aged healthy individuals and the frequency of TP53 mutations increases with age. 3–5 TP53 mutations rank top five among mutations identified in aged healthy individuals. 3–5 The TP53 gene, which encodes the tumorsuppressor p53, is the most frequent target for mutation in human cancer, including hematological malignancies. 6 TP53 mutations are present in 10% of MDS cases and 20–30% in secondary MDS arising after exposure to radiation or alkylating agents. 1, 7 The frequency of TP53 mutations in AML is approximately 10%. However, in AML with complex karyotype, the frequency of TP53 mutations and/or deletions is almost 70%. 8 Further, TP53 mutations are associated with poor prognosis and decreased survival in MDS and AML. 7–9 However, the role of TP53 mutations in clonal hematopoiesis and the pathogenesis of hematological malignancies is largely unknown. To date, no studies have investigated the in vivo effect of TP53 mutations expressed from the endogenous locus in hematopoietic cells, which might allow delineation of how these mutations contribute to the pathogenesis of hematological malignancies. 10-13 The vast majority of missense TP53 mutations are mapped to the DNA-binding domain of p53 protein and usually abrogate its sequence-specific DNA-binding activity. 6 Previous studies show that codon 248 of the p53 protein is most frequently mutated in MDS and AML. 7, 8 p53R248W mutant has also been identified in aged healthy individuals. 3–5 To better understand the role of mutant p53 in hematopoiesis when expressed physiologically from its endogenous promoter, we utilized the humanized p53R248W knock-in mice, expressing human p53 mutant protein from the endogenous murine Trp53 promoter. 14 Given that most TP53 mutations in clonal hematopoiesis and hematological malignancies are monoallelic missense mutations, 3–9 we examined HSC function in the heterozygous p53R248W/+ mice to recapitulate clinical conditions. We first analyzed the peripheral blood (PB) and bone marrow (BM) of p53+/+ and p53R248W/+ mice (8–12 week old). PB cell counts and BM cellularity were comparable among these mice (Supplementary Figures S1a–f). In p53 knockout mice, there is a dramatic increase of long-term HSCs11; however, we found that p53+/+ and p53R248W/+ mice have a similar number of long-term HSCs (Figure 1a and Supplementary Figure S1g). In addition, the expression of p53R248W did not affect the frequency of myeloid progenitors (Figures 1a and b, and Supplementary Figure S1g). To determine the role of p53R248W in hematopoiesis in vivo, we performed competitive BM transplantation assays as shown in Supplementary Figure S1h. We transplanted 5× 105 donor BM cells (p53+/+ or p53R248W/+, CD45. 2+) into lethally irradiated (9.5 Gy) recipient mice (CD45. 1+CD45. 2+) along with 5× 105 competitor marrow cells (CD45. 1+). At 16 weeks after transplantation, the repopulating ability of p53R248W/+ cells was significantly higher than that of the wild-type cells (Figure 1c). We also observed increased frequency of donor-derived HSPCs in the BM of recipient mice repopulated with p53R248W/+ cells (Figures 1d and e and Supplementary Figure S1i). p53R248W did not affect myeloid and lymphoid differentiation in the PB and the …