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Osteoclast-poor RANKL-dependent Autosomal Recessive Osteopetrosis (ARO) is a rare bone disease characterized by an increase in bone density due to the failure of bone resorption by impaired osteoclast formation. Hematopoietic stem cell transplantation is not an effective therapy for this ARO form, since in bone RANKL is produced mainly by cells of mesenchymal origin. Therefore Mesenchymal Stromal Cells (MSC) transplantation together with a gene-therapy strategy to correct RANKL defect in MSC could represent a possible effective therapy. Of note, whether also MSC, besides the osteoclasts, are affected by RANKL deficiency is unknown. To verify this, we established and characterized bone marrow derived MSC (BM-MSC) lines from the Rankl−/− (KO) mouse model, which recapitulates the human disease, and from wild type (WT) mice. No differences were found between KO and WT MSC in terms of morphology, immunophenotype and proliferation capacity. However, KO MSC displayed a reduced clonogenic potential with a decrease in stemness genes expression. KO MSC were able to normally differentiate towards the adipogenic and chondrogenic lineages, while showed a significantly impaired osteogenic differentiation capacity compared to WT MSC, as demonstrated by reduced Alizarin Red staining (ARS) and expression of osteogenic genes. To confirm that this alteration was due to the lack of functional RANKL, we developed a third generation lentiviral vector expressing human soluble RANKL (hsRL) for the genetic correction of KO MSC. We first investigated lentiviral transduction in 293T cells to optimize transduction efficiency at different multiplicity of infection (MOI) ranging from 1 to 100. hsRL production increased proportionally to the MOI and was stable over time. However, the higher the MOI the higher the cytotoxicity observed. Based on these data, we performed a lentiviral hsRL transduction in KO MSC at 20 and 50 MOI, to define the optimal transduction conditions. After transduction 99.5% of MSC were GFP+. While in Rankl−/− control cells the cytokine was not detected, in corrected cells hsRL production and secretion was measurable and comparable to sRL levels in WT mouse. KO MSC stably expressing hsRL showed an improved osteogenic differentiation capacity compared to untransduced KO MSC, as demonstrated by increased ARS and expression of osteogenic genes. Moreover, the expression of RANK receptor in both MSC suggested an autocrine role of sRL as possible mechanism. Our data suggest that restoration of RANKL production in lentiviral-transduced KO MSC might not only allow osteoclast differentiation in Rankl−/− mice upon transplantation, but also improve the osteogenic differentiation defect of KO MSC.