Lentivirus-based ex-vivo gene therapy for hematopoietic stem and progenitor cells (HSPCs) has shown impressive results, but genotoxic potential of insertional mutagenesis still raises safety concerns. Gene editing (GE) tools offer a safer alternative by enabling the selection of insertion sites for expression cassettes. Aiming to achieve high levels of transgene expression specifically in the myeloid lineage after HSPC editing, we targeted CX3CR1 4th intron using CRISPR-Cas9. Different AAV6 donor DNA templates containing a SFFV promoter and a GFP reporter gene were designed to optimise knock-in process. Surprisingly, specific targeted insertion lead to increased levels of CX3CR1 on HSPCs, but not in other cell types. Our data showed that the CX3CR1 increment was greater on the primitive CD34+CD4RA- population. But also, that the reporter followed a myeloid-skewed expression pattern, being the highest GFP expression on CD34- committed myeloid populations during their stem culture. This suggests that the integrated cassette is being down regulated or silenced due to the epigenetic state of CX3CR1 on early stages of HSCPs differentiation. Therefore, we propose that CX3CR1 can be used as a safe harbour for targeted insertion of expression cassettes on HSPCs, with the advantage of a safer profile on stem stages while promoting higher expression upon differentiation into promyelocytes.