Osteogenesis occurs in striking interaction with angiogenesis. There is growing evidence that endothelial cells are involved in the modulation of osteoblast differentiation. We hypothesized that primary human umbilical vein endothelial cells (HUVEC) should be able to modulate primary human osteoprogenitors (HOP) function in an in vitro co-culture model. In a previous study we demonstrated that a 3 day to 3 week co-culture stimulates HOP differentiation markers such as Alkaline Phosphatase (ALP) activity and mineralization. In the present study we addressed the effects induced by the co-culture on HOP within the first 48 hours. As a prerequisite, we validated a method based on immuno-magnetic beads to separate HOP from HUVEC after co-culture. Reverse transcription-real time quantitative PCR studies demonstrated up-regulation of the ALP expression in the co-cultured HOP, confirming previous results. Surprisingly, down-regulation of runx2 and osteocalcin was also shown. Western blot analysis revealed co-culture induced down-regulation of Connexin43 expression in both cell types. Connexin43 function may be altered in co-cultured HOP as well. Stimulation of the cAMP pathway was able to counterbalance the effect of the co-culture on the ALP activity, but was not able to rescue runx2 mRNA level. Co-culture effect on HOP transcriptome was analyzed with GEArray cDNA microarray showing endothelial cells may also modulate HOP extracellular matrix production. In accordance with previous work, we propose endothelial cells may support initial osteoblastic proliferation but do not alter the ability of the osteoblasts to produce extracellular mineralizing matrix.