<p>Human pluripotent stem (hPS) cell-derived blood-generating heart-forming organoids (BG-HFOs) represent a highly structured in vitro model that recapitulates aspects of human cardiac, hematoendothelial and multiwave hematopoietic co-development. Our model offers novel insights into human development and represents a potent tool for disease modeling, drug testing and other advanced in vitro assays. Here we provide a detailed 14-d protocol for the generation of BG-HFOs, from the embedding of hPS cell-derived aggregates in Matrigel, directing cell differentiation via WNT pathway modulation and supplementation of cytokine cocktails required for hematoendothelial induction and maturation. The protocol is robust and applicable to different hPS cell lines. Proper formation and patterning of the multiple tissues present in the BG-HFOs can be assessed through techniques such as live-cell imaging, whole-mount immunofluorescence (IF) staining, flow cytometry and gene-expression analysis. The efficient generation of BG-HFOs requires hands-on experience with hPS cell culture and the simultaneous management of multiple medium-enriching growth factors and small molecules. We additionally highlight a simple, scalable method of sample preparation for the effective investigation of BG-HFO morphology via laser microscopy using common laboratory equipment. This Protocol requires basic skills in handling hazardous chemicals and limited experience in IF staining and laser microscopy. Our robust and fast method enables the whole-mount IF staining and clearing of large organoids (up to 4 mm in diameter) composed of multiple tissues (with different physical properties) such as BG-HFOs and other complex organoid models in 2.5 d. The described workflow can thus promote fast laser microscopy, thereby tackling a major challenge in the field.</p>