Abnormal nuclear morphology is a hallmark of malignant cells widely used in cancer diagnosis. Pelger-Huët anomaly (PHA) is a common abnormality of neutrophil nuclear morphology of unknown molecular etiology in myeloid neoplasms (MNs). We show that loss of nuclear lamin B1 (LMNB1) encoded on chromosome 5q, which is frequently deleted in MNs, induces defects in nuclear morphology and human hematopoietic stem cell (HSC) function associated with malignancy. LMNB1 deficiency alters genome organization inducing in vitro and in vivo expansion of HSCs, myeloid-biased differentiation with impaired lymphoid commitment, and genome instability due to defective DNA damage repair. Nuclear dysmorphology of neutrophils in patients with MNs is associated with 5q deletions spanning the LMNB1 locus, and lamin B1 loss is both necessary and sufficient to cause PHA in normal and 5q-deleted neutrophils. LMNB1 loss thus causes acquired PHA and links abnormal nuclear morphology with HSCs and progenitor cell fate determination via genome organization. Display omitted •LMNB1 gene is commonly deleted in myeloid malignancies•Loss of LMNB1 promotes self-renewal and myeloid-biased hematopoiesis•Loss of LMNB1 causes acquired Pelger-Huët neutrophil nuclear anomaly•Loss of LMNB1 alters 3D genome organization in HSPCs and neutrophils Abnormal nuclear morphology is a hallmark of cancerous cells. Here, Reilly et al. demonstrate that deletion of lamin B1, which is common in myeloid malignancies, causes acquired Pelger-Huët nuclear anomaly and links aberrant nuclear morphology with HSC fate determination via 3D genome organization.