The human body is composed of about 3−4×1013 (30-40 trillion) cells [1]. These cells are all functioning consistently, and working elegantly together, to sustain the organism. Not only humans, but all other living things on earth (from plants to parrots) are composed of cells. Cells are the smallest living building blocks of plants and animals, and in fact some organisms are built from a single cell, such as bacteria. To be able to sustain life, cells are dynamic entities that need to grow, divide, and interact with their environment. They accomplish this by a number of complex and dynamic processes. For instance, to perform vital functions such as cell division, a key factor of life, cells need to dramatically change their shape. In addition to shape changes, the internal cellular organization needs to be tightly controlled to properly function. For example, cells need to establish a front-back polarity to drive directional migration, like immune cells that hunt for intruders. Furthermore, the proper functioning of brain cells (also named neurons), which depends on finding and connecting to other neuronal cells, is closely related to their internal organization and cellular shape. For cells, essentially small bags filled with proteins, to change their shape and internal organization, they depend on an internal filamentous scaffold named the cytoskeleton. Unlike the name might suggest, this ’cellular skeleton’ is actually very dynamic, with constant assembly and disassembly of the constituent filaments and changes in filament organization. In addition to organizing the cellular interior, this cytoskeleton provides mechanical support for cells and allows them to generate forces. Two main cytoskeletal components are microtubules and actin filaments. They are usually studied as separate systems, despite a growing body of work indicating their functions are closely intertwined and interdependent. This thesis studies how these two cytoskeletal components influence each other. More specifically, we focus on the question how actin and microtubules co-organize and affect each other via proteins that physically link them to each other, named cytolinkers. To study how cytolinkers impact cytoskeletal crosstalk, we move away from the complex environment of the cell, where many other proteins are present and different processes take place. We took the cytoskeletal building blocks and cytolinking proteins out of the cell, rebuilt a cytoskeleton from these building blocks and characterized the effects of the cytolinkers on cytoskeletal co-organization by fluorescence microscopy. In addition to natural cytolinkers, we engineered our own cytolinkers to better understand how these proteins influence microtubule/actin coordination and in the absence of illdefined regulatory processes in the cell. This isolated context is a powerful tool to study cellular functions, as the simplification allows us to tightly control all variables and identify the underlying mechanisms.