Located within red pulp cords, splenic red pulp macrophages (RPMs) are constantly exposed to the blood flow, clearing senescent red blood cells (RBCs) and recycling iron from hemoglobin. Here, we studied the mechanisms underlying RPM homeostasis, focusing on the involvement of stromal cells as these cells perform anchoring and nurturing macrophage niche functions in lymph nodes and liver. Microscopy revealed that RPMs are embedded in a reticular meshwork of red pulp fibroblasts characterized by the expression of the transcription factor Wilms' Tumor 1 (WT1) and colony stimulating factor 1 (CSF1). Conditional deletion of Csf1 in WT1+ red pulp fibroblasts, but not white pulp fibroblasts, drastically altered the RPM network without altering circulating CSF1 levels. Upon RPM depletion, red pulp fibroblasts transiently produced the monocyte chemoattractants CCL2 and CCL7, thereby contributing to the replenishment of the RPM network. Thus, red pulp fibroblasts anchor and nurture RPM, a function likely conserved in humans. Keywords: CSF1; colony stimulating factor 1; fibroblasts; homeostasis; macrophages; niche; spleen; stromal cells.