MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate various cellular processes, including cell differentiation and proliferation. This study identifies miR-145 and miR-143 as key regulators of smooth muscle cell fate decisions. These miRNAs are co-transcribed in cardiac progenitors and later localized to smooth muscle cells, including those derived from neural crest stem cells. They are direct targets of serum response factor (SRF), myocardin, and Nkx2.5, and are downregulated in injured or atherosclerotic vessels with proliferating, less differentiated smooth muscle cells. miR-145 is necessary for myocardin-induced reprogramming of adult fibroblasts into smooth muscle cells and sufficient to induce differentiation of neural crest stem cells into vascular smooth muscle. miR-145 and miR-143 cooperatively target a network of transcription factors, including Klf4, myocardin, and Elk-1, to promote differentiation and repress proliferation of smooth muscle cells. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of smooth muscle cells. The study also shows that miR-145 is necessary and sufficient for VSMC differentiation, and that miR-145 and miR-143 target a network of factors to promote VSMC differentiation and repress proliferation. The results suggest that miR-145 may promote VSMC differentiation by directly repressing numerous transcription factors that promote the proliferative state while stabilizing factors that promote the differentiated state of VSMCs. The study also highlights the potential therapeutic value of restoring normal levels of miR-143 and miR-145 in disease settings.MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate various cellular processes, including cell differentiation and proliferation. This study identifies miR-145 and miR-143 as key regulators of smooth muscle cell fate decisions. These miRNAs are co-transcribed in cardiac progenitors and later localized to smooth muscle cells, including those derived from neural crest stem cells. They are direct targets of serum response factor (SRF), myocardin, and Nkx2.5, and are downregulated in injured or atherosclerotic vessels with proliferating, less differentiated smooth muscle cells. miR-145 is necessary for myocardin-induced reprogramming of adult fibroblasts into smooth muscle cells and sufficient to induce differentiation of neural crest stem cells into vascular smooth muscle. miR-145 and miR-143 cooperatively target a network of transcription factors, including Klf4, myocardin, and Elk-1, to promote differentiation and repress proliferation of smooth muscle cells. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of smooth muscle cells. The study also shows that miR-145 is necessary and sufficient for VSMC differentiation, and that miR-145 and miR-143 target a network of factors to promote VSMC differentiation and repress proliferation. The results suggest that miR-145 may promote VSMC differentiation by directly repressing numerous transcription factors that promote the proliferative state while stabilizing factors that promote the differentiated state of VSMCs. The study also highlights the potential therapeutic value of restoring normal levels of miR-143 and miR-145 in disease settings.