Indirect co-culture, wherein two distinct cell types are cultivated within the same medium without direct contact, remains a relatively underexplored approach in biomaterials science for simulating physiological cell-cell interactions on material surfaces in vitro. In this study, human mesenchymal stem cells (hMSCs) were cultured on two types of Ti6Al4V substrates (polished and sand-blasted/acid etched) in a co-culture system using conditioned osteogenic differentiation media (cOBM), enriched with soluble factors secreted by human osteoblasts (hOBs). The combined impact of surface microtopography of Ti6Al4V substrates and cOBM supplementation has resulted in the modulation of cell morphology, alkaline phosphatase (ALP) activity, and calcium phosphate mineralization. Enhanced mineralization (2.5-fold increase compared to baseline at day 21) was observed on Ti6Al4V substrates when hMSCs were cultured in the presence of cOBM. This was accompanied by a peak expression of the early osteogenic marker, ALP by day 14. The synergistic behavior of sandblasted and acid-etched substrates with soluble biochemical cues, derived from hOBs showcased their potential for augmenting osteogenic differentiation. The in vitro outcomes were validated in a rabbit model study, which clearly demonstrated better osseointegration of sand-blasted/acid etched implants over 12 weeks.