Bacillus subtilis spore formation has been studied as a model for cellular differentiation, but recent research reveals that spore formation in surface-associated communities (biofilms) exhibits previously unknown spatial organization. Motile cells differentiate into aligned chains of attached cells that form aerial structures, or fruiting bodies, which serve as sites for sporulation. Fruiting body formation depends on regulatory genes involved in early sporulation and genes required for exopolysaccharide and surfactin production. Natural isolates of B. subtilis form robust pellicles with intricate structures, while laboratory strains do not, indicating that multicellularity has been lost during domestication. Other microbial differentiation processes may also display spatial organization when studied with natural isolates. Microbes develop complex pathways in response to environmental cues. B. subtilis spore formation is a well-studied process, involving the differentiation of a single cell into two types: the mother cell and the forespore. However, these studies have focused on unicellular organisms. In natural settings, microorganisms often exist as multicellular communities with high structural complexity. This study investigated B. subtilis biofilm formation, finding that natural isolates form structured pellicles, while laboratory strains do not. Mutants lacking key sporulation genes failed to form pellicles or produce spores. Genes like yveQ and yveR, involved in exopolysaccharide biosynthesis, are critical for pellicle formation and the development of aerial structures. Surfactin, a biosurfactant, is also important for pellicle formation and aerial structure development. The formation of aerial structures is a key site for sporulation, with spore-specific gene expression concentrated at the tips of these structures. This spatial organization is reminiscent of multicellular organisms. The study shows that B. subtilis forms spore-filled fruiting bodies in surface-associated communities, which could facilitate spore dispersal. The results indicate that sporulation in B. subtilis is a social process with high temporal and spatial organization. This challenges the traditional view of bacteria as unicellular organisms. The study highlights the importance of structured communities in microbial development and suggests that many microbial processes previously thought to be unicellular may exhibit multicellular features when studied in structured environments like biofilms.Bacillus subtilis spore formation has been studied as a model for cellular differentiation, but recent research reveals that spore formation in surface-associated communities (biofilms) exhibits previously unknown spatial organization. Motile cells differentiate into aligned chains of attached cells that form aerial structures, or fruiting bodies, which serve as sites for sporulation. Fruiting body formation depends on regulatory genes involved in early sporulation and genes required for exopolysaccharide and surfactin production. Natural isolates of B. subtilis form robust pellicles with intricate structures, while laboratory strains do not, indicating that multicellularity has been lost during domestication. Other microbial differentiation processes may also display spatial organization when studied with natural isolates. Microbes develop complex pathways in response to environmental cues. B. subtilis spore formation is a well-studied process, involving the differentiation of a single cell into two types: the mother cell and the forespore. However, these studies have focused on unicellular organisms. In natural settings, microorganisms often exist as multicellular communities with high structural complexity. This study investigated B. subtilis biofilm formation, finding that natural isolates form structured pellicles, while laboratory strains do not. Mutants lacking key sporulation genes failed to form pellicles or produce spores. Genes like yveQ and yveR, involved in exopolysaccharide biosynthesis, are critical for pellicle formation and the development of aerial structures. Surfactin, a biosurfactant, is also important for pellicle formation and aerial structure development. The formation of aerial structures is a key site for sporulation, with spore-specific gene expression concentrated at the tips of these structures. This spatial organization is reminiscent of multicellular organisms. The study shows that B. subtilis forms spore-filled fruiting bodies in surface-associated communities, which could facilitate spore dispersal. The results indicate that sporulation in B. subtilis is a social process with high temporal and spatial organization. This challenges the traditional view of bacteria as unicellular organisms. The study highlights the importance of structured communities in microbial development and suggests that many microbial processes previously thought to be unicellular may exhibit multicellular features when studied in structured environments like biofilms.