Mural cells (MCs) are essential for blood vessel stability and function; however, the mechanisms that regulate MC development remain incompletely understood, in particular those involved in MC specification. Here, we investigated the first steps of MC formation in zebrafish using transgenic reporters. Using and reporters, we show that the onset of expression of , a pericyte marker in adult mice and zebrafish, occurs almost coincidentally with an increment in expression in peri-arterial mesenchymal cells, suggesting that these transcriptional changes mark the specification of MC lineage cells from naïve mesenchymal cells. The emergence of peri-arterial MCs required Notch signaling. We found that -positive cells express in addition to , and although depletion of or failed to block MC emergence, embryos depleted of both and lost mesoderm- as well as neural crest-derived MCs. Using reporters that read out Notch signaling and Notch2 receptor cleavage, we show that Notch activation in the mesenchyme precedes specification into MCs. Taken together, these results show that Notch signaling is necessary for peri-arterial MC specification.