Erythroid island (EI) niche are structures in the bone marrow and spleen where erythroblasts proliferate and differentiate prior to entering circulation as fully matured red blood cells (RBCs). Macrophages work as the core cells within EI niches, and previous studies show that, via adhesion molecules and secreted factors, they support adult erythropoiesis assisting mainly in haemoglobin synthesis and engulfment of the erythroblasts’ extruded nuclei. We have developed an in vitro approach to study the EI niche, where genetically modified macrophages are generated from the iKLF1-iPSC line, where KLF1 is activated upon tamoxifen induction. KLF1-activated macrophages were able to mimic the EI-niche microenvironment in vitro - when in culture with CD34+ umbilical cord blood cells, a higher number of mature RBCs were produced compared to cultures with wild type macrophages. RNA sequencing of KLF1-activated macrophages identified a high number of EI niche related genes, together with secreted factors responsible for RBC maturation. To further understand which proteins are associated with macrophage-erythroblast communication and maturation in the EI niche we carried a proteomics analysis on KLF1-activated macrophages. The study of the proteome of these cells showed that most upregulated proteins are associated with cytoskeleton and cellular membrane and are responsible for their cellular organization and signalling activity. Extracellular membrane vesicles related protein RAB27b is also highly regulated on KLF1-activated macrophages. The potential for RBC maturation of other upregulated proteins in KLF1-activated macrophages – TRFC, LAMP2 and LMAN1 - was also tested. The study of these protein in the context of the EI-niche microenvironment could uncover novel mechanisms associated with the interactions between macrophages and erythroid cells during adult erythropoiesis. Erythroid island (EI) niche are structures in the bone marrow and spleen where erythroblasts proliferate and differentiate prior to entering circulation as fully matured red blood cells (RBCs). Macrophages work as the core cells within EI niches, and previous studies show that, via adhesion molecules and secreted factors, they support adult erythropoiesis assisting mainly in haemoglobin synthesis and engulfment of the erythroblasts’ extruded nuclei. We have developed an in vitro approach to study the EI niche, where genetically modified macrophages are generated from the iKLF1-iPSC line, where KLF1 is activated upon tamoxifen induction. KLF1-activated macrophages were able to mimic the EI-niche microenvironment in vitro - when in culture with CD34+ umbilical cord blood cells, a higher number of mature RBCs were produced compared to cultures with wild type macrophages. RNA sequencing of KLF1-activated macrophages identified a high number of EI niche related genes, together with secreted factors responsible for RBC maturation. To further understand which proteins are associated with macrophage-erythroblast communication and maturation in the EI niche we carried a proteomics analysis on KLF1-activated macrophages. The study of the proteome of these cells showed that most upregulated proteins are associated with cytoskeleton and cellular membrane and are responsible for their cellular organization and signalling activity. Extracellular membrane vesicles related protein RAB27b is also highly regulated on KLF1-activated macrophages. The potential for RBC maturation of other upregulated proteins in KLF1-activated macrophages – TRFC, LAMP2 and LMAN1 - was also tested. The study of these protein in the context of the EI-niche microenvironment could uncover novel mechanisms associated with the interactions between macrophages and erythroid cells during adult erythropoiesis.