Altered splicing contributes to the pathogenesis of human blood disorders including myelodysplastic syndromes (MDS) and leukemias. Here we characterize the transcriptomic regulation of PRPF40B, which is a splicing factor mutated in a small fraction of MDS patients. We generated a full PRPF40B knockout (KO) in the K562 cell line by CRISPR/Cas9 technology and rescued its levels by transient overexpression of wild-type (WT), P383L or P540S MDS alleles. Using RNA sequencing, we identified hundreds of differentially expressed genes and alternative splicing (AS) events in the KO that are rescued by WT PRPF40B, with a majority also rescued by MDS alleles, pointing to mild effects of these mutations. Among the PRPF40B-regulated AS events, we found a net increase in exon inclusion in the KO, suggesting that this splicing factor primarily acts as a repressor. PRPF40B-regulated splicing events are likely cotranscriptional, affecting exons with A-rich downstream intronic motifs and weak splice sites especially for 5′ splice sites, consistent with its PRP40 yeast ortholog being part of the U1 small nuclear ribonucleoprotein. Loss of PRPF40B in K562 induces a KLF1 transcriptional signature, with genes involved in iron metabolism and mainly hypoxia, including related pathways like cholesterol biosynthesis and Akt/MAPK signaling. A cancer database analysis revealed that PRPF40B is lowly expressed in acute myeloid leukemia, whereas its paralog PRPF40A expression is high as opposed to solid tumors. Furthermore, these factors negatively or positively correlated with hypoxia regulator HIF1A, respectively. Our data suggest a PRPF40B role in repressing hypoxia in myeloid cells, and that its low expression might contribute to leukemogenesis.