A network of molecular factors drives the development, differentiation, and maintenance of endothelial cells. Friend leukemia integration 1 transcription factor (FLI1) is a bona fide marker of endothelial cells during early development. In zebrafish Tg( f li1:EGFP) y1 , we identified two endothelial cell populations, high-fli1 + and low-fli1 +, by the intensity of green fluorescent protein signal. By comparing RNA-sequencing analysis of non-fli1 expressing cells (fli1 -) with these two (fli1 +) cell populations, we identified several up-regulated genes, not previously recognized as important, during endothelial development. Compared with fli1 - and low-fli1 + cells, high-fli1 + cells showed up-regulated expression of the zinc finger transcription factor PRDI-BF1 and RIZ homology domain containing 16 (prdm16). Prdm16 knockdown (KD) by morpholino in the zebrafish larva was associated with impaired angiogenesis and increased number of low-fli1 + cells at the expense of high-fli1 + cells. In addition, PRDM16 KD in endothelial cells derived from human-induced pluripotent stem cells impaired their differentiation and migration in vitro. Moreover, zebrafish mutants (mut) with loss of function for the oncogene LIM domain only 2 (lmo2) also showed reduced prdm16 gene expression combined with impaired angiogenesis. Prdm16 expression was reduced further in endothelial (CD31+) cells compared with CD31- cells isolated from l mo2-mutants (l mo2-mut) embryos. Chromatin immunoprecipitation-PCR demonstrated that Lmo2 binds to the promoter and directly regulates the transcription of prdm16 This work unveils a mechanism by which prdm16 expression is activated in endothelial cells by Lmo2 and highlights a possible therapeutic pathway by which to modulate endothelial cell growth and repair.