RNA-binding proteins (RBPs) integrate the processing of RNAs into post-transcriptional gene regulation, but the direct contribution of them to myeloid cell specification is poorly understood. Here, we report the first global RBP transcriptomic analysis of myeloid differentiation by combining RNA-seq analysis with myeloid induction in CD34+ hematopoietic progenitor cells. The downregulated expression of the KH-Type Splicing Regulatory Protein (KSRP) during monocytopoiesis and up-regulated expression during granulopoiesis suggests that KSRP has divergent roles during monocytic and granulocytic differentiation. A further comparative analysis of miRNA transcripts reveals that KSRP promotes the biogenesis of miR-129, and the expression patterns and roles of miR-129 in myeloid differentiation are equivalent to those of KSRP. Finally, miR-129 directly blocks the expression of Runt Related Transcription Factor 1 (RUNX1), which evokes transcriptional modulation by RUNX1. Based on our findings, KSRP, miR-129, and RUNX1 participate in a regulatory axis to control the outcome of myeloid differentiation.