The study was designed to establish the biological basis for the use of autologous bone-marrow stromal cells (MSC) in order to improve the curing opportunities of congenital pseudarthrosis of the tibia (CPT). The investigation was planned by taking into account that the pathophysiology of bone healing mainly depends on the osteogenic potential of the resident cells, although several factors play a crucial role in restoring the normal bone structure. Bone marrow samples were collected from the lesion site (P) and the iliac crest (IC) of 7 patients affected by CPT and type 1 neurofibromatosis (NF1+) and 6 patients affected by CPT without NF1 (NF1-). Four patients without CPT served as control group. Biochemical, functional and molecular assays showed that the ability to generate bone-forming cells was higher in IC-MSC than in P-MSC, but lower in CPT patients than in control group. We evaluated whether host factors, such as autologous serum and the microenvironment surrounding the pseudarthrosis lesion, could impair the osteogenic differentiation of IC-MSC. Autologous serum was less effective than FBS in promoting the IC-MSC differentiation, but the damage was more evident in NF1- than in NF1+ patients. Additionally, the supernatant of osteoblast cultures obtained from bone fragments close to the lesion site favoured the differentiation of IC-MSC in NF1- patients. In summary, our results suggest that MSC transplantation could be a promising strategy for the therapy of CPT. Further studies are warranted to confirm the clinical effectiveness in comparison to standard surgical treatment.