We report that simultaneous expression of Akt and angiopoietin-1 (Ang-1) transgenes supported mitogenesis in stem cells with a critical role for microRNA-143 (miR-143) downstream of FoxO1 transcription factor. Mesenchymal stem cells (MSC) from young male rats were transduced with Ad-vectors encoding for Akt ((Akt)MSC) and Ang-1 ((Ang-1)MSC) transgenes for their individual or simultaneous overexpression ((AA)MSC; > 5-fold gene level and > 4-fold Akt and Ang-1 protein expression in (AA)MSC vs. Ad-Empty transduced MSC; (Emp)MSC). (AA)MSC had higher phosphorylation of FoxO1, which activated Erk5, a distinct mitogen-induced MAPK that drove transcriptional activation of cyclin D1 and Cdk4. Flow cytometry showed > 10% higher S-phase cell population that was confirmed by BrdU assay (15%) and immunohistology for Ki67 (11%) in (AA)MSC using (Emp)MSC as controls. miR array supported by real-time PCR showed induction of miR-143 in (AA)MSC (4.73-fold vs.. (Emp)MSC). Luciferase assay indicated a dependent relationship between miR-143 and Erk5 in (AA)MSC. FoxO1-specific siRNA upregulated miR-143, whereas inhibition of miR-143 did not change FoxO1 activation. However, miR-143 inhibition repressed phosphorylation of Erk5 and abrogated cyclin D1 with concomitant reduction in cells entering cell cycle. During in vivo studies, male GFP+ (AA)MSC transplanted into wild-type female infarcted rat hearts showed significantly higher number of Ki67 expressing cells (p < 0.05 vs. (Emp)MSC) 7 days after engraftment (n = 4 animals/group). In conclusion, co-overexpression of Akt and Ang-1 in MSC activated cell cycle progression by upregulation of miR-143 and stimulation of FoxO1 and Erk5 signaling.