Expansion of human megakaryoblasts from peripheral blood-derived CD34 + cells is commonly used to characterize inherited or acquired thrombocytopenia and evaluate defects in megakaryocyte (MK) differentiation, MK maturation and proplatelet formation. We applied single-cell RNA sequencing to understand local gene expression changes during megakaryopoiesis (days 6 and 11 of differentiation) in peripheral CD34 + cells from healthy controls and patients with ETV6 -related thrombocytopenia. Analysis of gene expression and regulon activity revealed distinct clusters partitioned into seven major cell stages: hematopoietic stem/progenitor cells (HSPC), common-myeloid progenitors (CMP), MK-primed CMP, granulocyte-monocyte progenitors, megakaryocyte-erythroid progenitors (MEP), MK progenitor /mature MK (MKP/MK) and platelets. We observed a subpopulation of MEP that arose directly from HSPC, deviating from the canonical MK differentiation pathway. ETV6 deficiency was characterized by an increase in HSPC, a decrease in MKP/MK, and a lack of platelets. ETV6 deficiency also led to the development of aberrant MEP and MKP/MK cell populations. Genes involved in “mitochondrial” and “DNA repair” pathways were downregulated, while genes involved in “translation” pathways were upregulated. Analysis of patient samples and hematopoietic cell lines transduced with an ETV6 variant revealed increased translation in MK. Ribosomal protein small 6 (RPS6) levels in MK, platelets and peripheral blood mononuclear cells was consistent with the translation findings. Our results provide a framework to understand peripheral CD34 + cell-derived megakaryocytic cultures. Our observations also shed light on ETV6 -variant pathology and reveal potential targets for diagnostic and therapeutic purposes. Key points - scRNAseq gain insight into in vitro megakaryopoiesis, identify MK-primed CMP, and a differentiation trajectory that bypasses the CMP. - ETV6 variants led to the development of aberrant MEP and MK cell populations.