Kilayim, Chapter 2, Mishnah 6. The Mishnah opens: "HaRotzeh la'asos sadeihu meitzar meitzar mikol min" - one who wishes to plant his field in narrow strips, row after row of different species, must make sure that there is a separation between the rows. How large must that separation be? On this Beis Shammai and Beis Hillel disagree.
The dispute between Beis Shammai and Beis Hillel regarding the size of the separation:
"Beis Shammai omrim: sheloshah telamim shel pasiach" - three furrows of the type called 'pasiach', which are the furrows that were generally made in order to break open the ground at the outset.
"U'Beis Hillel omrim: melo ha'ol haSharoni" - the measure of the full length of the yoke that was commonly used in the region called Sharon.
These two measurements are not precisely known to us today, but the Mishnah adds: "U'kerovin divrei eilu l'divrei eilu" - the opinions of Beis Shammai and Beis Hillel are very close to one another, so much so that it makes no practical difference which of them we follow.
The practical measure - two amos by two amos:
The Mishnah explains and provides a measure that can be used: the gap between the rows must be two amos wide and two amos long. Once there is a square of two amos by two amos, the two species may be brought very close to one another. As long as there is a significant gap of two amos by two amos separating between the rows, then before those two amos and after them the species may be brought right up against each other, since that square supplies the necessary distinction which makes clear that they are not being planted in a jumble.
And as we learned earlier, since these matters are rabbinic, on account of how things appear to the eye, as long as there is something that makes clear that the species are not being planted together entirely, they may be close to one another.
In summary: that box of two amos by two amos is all that is required in order to maintain the separation of the different species from one another.