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Simple Summary: The cartilage-to-bone transition is an essential process in healthy bone development and repair. Our previous work has shown that when the cells found within the human periosteum (the membrane surrounding the bone) are cultured in human serum (HS) as opposed to the standard animal serum (FBS), these cells have greater bone-forming capacity as assessed in an ectopic assay in nude mice. What is not understood is the molecular interactions that permitted this enhanced biological potency. Herein, virtual networks are created to identify the key proteins driving increased bone formation from these cells. Key signalling factors were identified through a network analysis, where FGFR3 was pinpointed as a major differential regulator between cells grown in HS and cells grown in FBS. This analysis was validated through an analysis of human-derived periosteal progenitor cells (PDCs) containing a constitutively active (ca) FGFR3. Following removal and analysis, we found that the FGFR3-ca cells that were implanted on bone void filler scaffolds in mice had an abundance of bone and cartilage that were present compared to the scaffold containing normal/healthy cells. This suggests that these cells were undergoing enhanced cartilage-to-bone transitions and that this protein may be a potentially novel therapeutic target for diseases where the cartilage-to-bone transition is affected such as during poor fracture healing. Human periosteum-derived progenitor cells (hPDCs) have the ability to differentiate towards both the chondrogenic and osteogenic lineages. This coordinated and complex osteochondrogenic differentiation process permits endochondral ossification and is essential in bone development and repair. We have previously shown that humanised cultures of hPDCs enhance their osteochondrogenic potentials in vitro and in vivo; however, the underlying mechanisms are largely unknown. This study aimed to identify novel regulators of hPDC osteochondrogenic differentiation through the construction of miRNA-mRNA regulatory networks derived from hPDCs cultured in human serum or foetal bovine serum as an alternative in silico strategy to serum characterisation. Sixteen differentially expressed miRNAs (DEMis) were identified in the humanised culture. In silico analysis of the DEMis with TargetScan allowed for the identification of 1503 potential miRNA target genes. Upon comparison with a paired RNAseq dataset, a 4.5% overlap was observed (122 genes). A protein–protein interaction network created with STRING interestingly identified FGFR3 as a key network node, which was further predicted using multiple pathway analyses. Functional analysis revealed that hPDCs with the activating mutation FGFR3N540K displayed increased expressions of chondrogenic gene markers when cultured under chondrogenic conditions in vitro and displayed enhanced endochondral bone formation in vivo. A further histological analysis uncovered known downstream mediators involved in FGFR3 signalling and endochondral ossification to be upregulated in hPDC FGFR3N540K-seeded implants. This combinational approach of miRNA-mRNA-protein network analysis with in vitro and in vivo characterisation has permitted the identification of FGFR3 as a novel mediator of hPDC biology. Furthermore, this miRNA-based workflow may also allow for the identification of drug targets, which may be of relevance in instances of delayed fracture repair. [ABSTRACT FROM AUTHOR]