Hematopoiesis represents a meticulously regulated and dynamic biological process. Genetic aberrations affecting blood cells, induced by various factors, frequently give rise to hematological tumors. These instances are often accompanied by a multitude of abnormal post-transcriptional regulatory events, including RNA alternative splicing, RNA localization, RNA degradation, and storage. Notably, post-transcriptional regulation plays a pivotal role in preserving hematopoietic homeostasis. The DEAD-Box RNA helicase genes emerge as crucial post-transcriptional regulatory factors, intricately involved in sustaining normal hematopoiesis through diverse mechanisms such as RNA alternative splicing, RNA modification, and ribosome assembly. This review consolidates the existing knowledge on the role of DEAD-box RNA helicases in regulating normal hematopoiesis and underscores the pathogenicity of mutant DEAD-Box RNA helicases in malignant hematopoiesis. Emphasis is placed on elucidating both the positive and negative contributions of DEAD-box RNA helicases within the hematopoietic system.