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The evolutionary transition to complex multicellularity in the animal stem lineage remains a profound mystery. To gain insights into this puzzle, I focused on choanoflagellates, the closest living relatives of animals. Despite their significance, understanding of the environmental factors influencing choanoflagellate evolution, life history, and behaviors remains limited. My doctoral research focused on the characterization of Choanoeca flexa, a recently discovered colonial choanoflagellate. This enigmatic microorganism is an example of how, by exploring the diversity of choanoflagellates, we can uncover novel biological phenomena that help us reconstruct the biology of animal progenitors and better understand the origins of animals.Chapter 1 reviews the history of C. flexa as an emerging model organism. C. flexa forms monolayered colonies capable of reversible inversions between flagella-in and flagella-out conformations, each with distinct multicellular behaviors. The colony inversion of C. flexa mirrors aspects of embryogenesis and behavioral escape responses in animals. Thus, this system offers a unique platform to investigate the evolutionary mechanisms underlying the origins of animal multicellular development and behavior. Additionally, its reliable presence in splash pools of the Caribbean island of Curaçao provides an opportunity to study its ecology, making C. flexa an appealing and promising model for eco- and evo-cell biology. For the work in Chapter 2, I collaborated with Nuria Ros-Rocher (Pasteur Institute) to characterize the life history of C. flexa and its environmental influences. We discovered that C. flexa exhibits ‘clonal-aggregative development’, forming colonies through clonal division, aggregation, or a combination of both. Field and laboratory investigations revealed that C. flexa multicellularity is entrained by extreme salinity fluctuations typical of splash pools undergoing evaporation-refilling cycles. Upon gradual evaporation, colonies dissociate into solitary desiccation-resistant cells, which reform colonies after rehydration. We proposed that C. flexa clonal-aggregative development may offer a rapid and versatile route to multicellularity, which can be particularly advantageous in ephemeral habitats like splash pools. This discovery blurs the line between clonal and aggregative multicellularity and expands our notion about the evolutionary trajectory leading to animals. Finally, in Chapter 3, I demonstrate how studying choanoflagellate multicellular behavior can shed light on ancient animal signaling pathways. Specifically, I explored nitric oxide (NO) signaling in choanoflagellates and found that C. flexa and two other species express genes for canonical animal NO signaling. In C. flexa, NO induces colony inversion, partially mediated by the secondary messenger cGMP, similar to animals. Moreover, the behavioral output of NO signaling in C. flexa – contraction and regulation of feeding and swimming – parallels the effect of NO in certain animal groups. This discovery suggests that NO/cGMP signaling predates animals and may have regulated similar behaviors in their ancestors. Together, these findings highlight the potential of choanoflagellates, particularly C. flexa, as a valuable system for unraveling the mysteries of multicellularity and animal evolution.