Hematopoietic cells (HCs) and endothelial cells (ECs) can be produced in vitro from human embryonic stem cells (hESCs), but the differentiation systems used are still inefficient. To overcome this obstacle, it is necessary to understand the differentiation process. One of the methods used to obtain HCs and ECs from hESCs is their co-culture with stromal cells. The soluble factors secreted by these cells and cell-cell contact have a great impact on the differentiation process. Here, we performed comparative proteomic analyses of proteins obtained from the total extract of OP9 stromal cells and secreted by these cells before and during in vitro generation of HCs and ECs (hematoendothelial) from hESCs. We identified a total of 83 secreted and 759 intracellular proteins during differentiation. Twenty-five secreted and 181 proteins from the total extract were more abundant. Some secreted proteins are involved in cell-matrix interactions and HC and/or EC development. Moreover, 13 proteins of the total extract from OP9 cells that were exclusive/or more abundant during differentiation are involved in the Nrf2/Nfe2l2 gene pathway, that is, they are described to have a key role in oxidative stress and in hematopoietic development and maturation. Our proteomic profiles provide valuable insight about the proteins involved in in vitro hematoendothelial cell generation and in the future they might be used to optimize the differentiation process and produce both cell types in vitro.