Low-level expression of human factor VIII (fVIII) has limited the success of clinical trials for hemophilia A using both ex vivo and in vivo gene transfer methods. Ex vivo genetic modification provides increased control of gene transfer and permits thorough characterization of transgene copy number, chromosomal integration site(s), and expression levels prior to transplantation. A subpopulation of adherent bone marrow-derived cell types, termed mesenchymal stem cells (MSCs), comprise an attractive target cell type for ex vivo gene transfer due to their accessibility, ability to differentiate into multiple cell types, and long-term survival following transplantation. Recently we demonstrated, in vitro, that recombinant B-domain-deleted porcine fVIII (rp-fVIII) is expressed at levels 10 – 14-fold greater than recombinant B-domain-deleted human fVIII (rh-fVIII) due to an enhanced rate of secretion from baby hamster kidney-derived (BHK-M) cells. Additionally we found that only the A1 and activation peptide-A3 domain sequences of porcine fVIII are necessary to retain high-level expression of hybrid human/porcine fVIII constructs. Here we report the expression of rp-fVIII from murine MSCs isolated from exon-16 fVIII knockout mice following ex vivo retroviral transduction. MSCs were transduced with ecotropic envelope-pseudotyped murine stem cell virus containing a rp-fVIIII transgene at an multiplicity of infection of 2 – 5 functional viral particles/target MSC. Following this transduction regimen the cell population harbored an average of 1.8 proviral genomes/cell as determined by quantitative real-time PCR. During culture in growth medium supplemented with 20% fetal bovine serum (FBS) rp-fVIII-transduced MSCs demonstrated a steady-state level of 2,400 fVIII mRNA transcripts/cell and an apparent fVIII production rate of 174 units/106 cells/24 hr as determined by quantitative real-time RT-PCR and one-stage clotting assay, respectively. However, when cultured in serum-free medium the mRNA levels decreased to 950 transcripts/cell and apparent fVIII production was reduced to 14 units/106 cells/24 hr. The latter mRNA/fVIII expression ratio is in agreement with that previously reported from stably transduced BHK-M clonal cell lines. In contrast the former mRNA levels are lower than predicted from the observed fVIII activity levels. The activation quotient (ratio of apparent fVIII activity following thrombin pre-treatment to non-thrombin treated baseline fVIII activity) increased 17-fold when the cells were cultured in serum-free versus serum-containing medium. Additionally, SDS-PAGE analysis of immunoprecipitated fVIII protein from serum-containing and serum-free medium revealed the presence of activated fVIII (fVIIIa) in conditioned medium samples containing FBS. Highly purified rp-fVIII from transduced-MSCs cultured in serum-free medium displayed similar relative mobility to BHK-M produced rp-fVIII upon SDS-PAGE analysis. These data warn against the determination of fVIII activity in serum-containing medium using the one-stage coagulation assay due to the presence of activated fVIII with high specific activity. Based on these results it is reasonable to predict that hemophilia A could be cured using high-level expression fVIII constructs by transplantation of a feasible number (106 - 108) of cells containing a single copy of rp-fVIII or other high-level expression hybrid human/porcine fVIII transgenes.