Mesenchymal stromal cells (MSCs) are a key component of the bone marrow (BM) niche, providing essential support required for the maintenance of hematopoietic stem cells. To advance our understanding of physiological functions of p53 and Mdm2 in BM-MSCs, we developed traceable conditional mouse models targetingMdm2and/orTrp53in vivo. We demonstrate that Mdm2 is essential for the emergence, maintenance, and hematopoietic support of BM-MSCs.Mdm2haploinsufficiency in BM-MSCs resulted in genotoxic stress–associated thrombocytopenia, suggesting a functional role for Mdm2 in hematopoiesis. In a syngeneic mouse model of acute myeloid leukemia (AML),Trp53deletion in BM-MSCs improved survival, and protected BM against hematopoietic toxicity from a murine Mdm2i, DS-5272. The transcriptional changes were associated with dysregulation of glycolysis, gluconeogenesis, and Hif-1α in BM-MSCs. Our results reveal a physiologic function of Mdm2 in BM-MSC, identify a previously unknown role of p53 pathway in BM-MSC–mediated support in AML and expand our understanding of the mechanism of hematopoietic toxicity of MDM2is.