Prime Editing (PE) is a versatile CRISPR/Cas9-mediated gene editing tool that, unlike conventional strategies, functions as a "search and replace" genome editing method without the need of double-strand breaks in the DNA and a donor DNA template. Chromosome instability caused by improper DNA interstrand crosslink repair characterizes Fanconi anemia (FA) cells. Because of this deficiency, precise changes in the genome aiming the specific correction of disease-causing mutations opens new therapeutic opportunities to correct FA mutations in hematopoietic stem and progenitor cells (HSPCs) from FA patients. To study the possibility of targeting HSPCs by PE, primary human healthy donor CD34⁺ cells purified from cord blood (CB) and mobilized peripheral blood (mPB) cells were targeted using an optimized PEmax architecture, together with a control engineered prime editing guide RNA (epegRNA) that targets FANCF. In order to improve PE outcomes, we also used a nicking guide RNA that targets the non-edited strand (PE3 system). Our results showed editing efficacies of 21.0% and 20.86% in CD34⁺ cells derived from CB and mPB, respectively. Additionally, we optimized the PEmax mRNA in vitro transcription resulting in on-target editing rates up to 28.0% and 30.1% respectively, with almost no indels formation (less than 1%). Importantly, we also demonstrated that PE efficiently targets primitive HSCs as identified by flow cytometry. Finally, we demonstrated that edited CD34+ cells maintain their repopulation ability in immunodeficient NSG mice, confirming the relevance of PE to correct disorders affecting the hematopoietic system.