Bone is a dynamic tissue and adapts its architecture in response to biological and mechanical factors. Here we investigate how cortical bone formation is spatially controlled by the local mechanical environment in the murine tibia axial loading model (C57BL/6). We obtained 3D locations of new bone formation by performing ‘slice and view’3D ﬂuorochrome mapping of the entire bone and compared these sites with the regions of high ﬂuid velocity or strain energy density estimated using a ﬁnite element model, validated with ex-vivo bone surface strain map acquired ex-vivo using digital image correlation. For the comparison, 2D maps of the average bone formation and peak mechanical stimulus on the tibial endosteal and periosteal surface across the entire cortical surface were created. Results showed that bone formed on the periosteal and endosteal surface in regions of high ﬂuid ﬂow. Peak strain energy density predicted only the formation of bone periosteally. Understanding how the mechanical stimuli spatially relates with regions of cortical bone formation in response to loading will eventually guide loading regime therapies to maintain or restore bone mass in speciﬁc sites in skeletal pathologies.