The primary walls of plant cells contain abundant levels of xyloglucan (XyG), a structurally complex collection of polysaccharides whose backbone consists of β-1,4-linked glucosyl residues that are generally highly substituted by xylosyl residues and other sugars. While the structure of XyG has been deciphered, its biosynthesis remains yet to be fully clarified. For example, it is well established that the loss of genes encoding XyG xylosyltransferases (XXT genes) lack detectable levels of XyG but are phenotypically relatively normal under standard growth conditions. It is also known that the nasturtium CSLC gene and an Arabidopsis CSLC4 gene produce short β-1,4-linked glucan chains when expressed in the yeast Pichia. When co-expressed in Pichia, CSLC4 and XXT1 produce long chains of β-1,4-linked glucan. Despite these findings, there is yet a lack of in vivo evidence for a role of the Arabidopsis CSLC family members as β-1,4-linked glucan synthases. It is also unknown how plants devoid XyG exhibit no visible phenotypical alteration. We addressed these gaps using single and higher order mutants of the Arabidopsis CSLC family members. We established that a quintuple mutant with disruptions in all five CSLC genes had no detectable XyG and that it did not exhibit drastic phenotypes in development and gene expression compared to wild type. Furthermore, we demonstrated that each of the five CSLC genes could complement the lack of XyG production of the quintuple mutant, supporting that each of them encodes a XyG glucan synthase. Therefore, our results demonstrate a role of the CSLC genes in XyG biosynthesis in vivo and provide new tools to the community to address the role of XyG in plant cell wall structure and function.