In recent years, genes encoding components of the RNA splicing machinery and regulatory factors have been characterized as a major class of mutated genes in myelodysplastic syndromes (MDS), with mutations occurring in a heterozygous gain‐of‐function pattern at recurrent amino acids. However, the effects on splicing patterns and consequent pathogenesis of malignancy remain poorly understood, in part due to challenges in isolating appropriate homogenous cellular populations carrying these mutations. We employed two complementary approaches to more effectively study splicing factors mutated in myeloid malignancies. We developed protocols to isolate bone marrow hematopoietic stem cells or peripheral blood progenitor cells from MDS patients known to carry splicing factor mutations. Our goal was to perform single‐cell whole transcriptome RNA sequencing and compare differences in splicing between mutated and non‐mutated cells from the same patient, as well as between patients. We also created genome‐edited cell lines, using the CRISPR/Cas system to alter single DNA base pairs in cell lines to reproduce the mutations found in patients. We successfully generated K562 cell lines with mutations in SRSF2 and U2AF1, two of the most frequently mutated splicing factors. Importantly, these cell lines demonstrate the viability of this novel technology in hematopoietic cells as well as provide effective tools to study the molecular consequences of splicing factor mutations. Together, these approaches will allow better characterization of the role of splicing factors in the pathogenesis of myeloid malignancies.